Anti-neoplastic combinations and dosing regimens using cdk4/6 inhibitor compounds to treat rb-positive tumors

ABSTRACT

This invention directed to methods for treating select RB-positive cancers and other Rb-positive abnormal cellular proliferative disorders using CDK4/6 inhibitors in specific dosing and combination or alternation regimes. In one aspect, treatments of select RB-positive cancers are disclosed using specific CDK4/6 inhibitors in combination or alternation with a Bruton&#39;s tyrosine kinase (BTK) inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/254,364, filed Jan. 22, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/457,699, filed Mar. 13, 2017, which is acontinuation of International Patent Application No. PCT/US2015/049777,filed Sep. 11, 2015, which claims the benefit of U.S. ProvisionalApplication No. 62/050,043, filed Sep. 12, 2014. The entirety of each ofthese applications are hereby incorporated by reference for allpurposes.

FIELD

This invention is in the area of methods for treating select RB-positivecancers and other Rb-positive abnormal cellular proliferative disordersusing the described CDK4/6 inhibitors in specific dosing and combinationor alternation regimes. In one aspect, treatments of select RB-positivecancers are disclosed using specific CDK4/6 inhibitors in combination oralternation with another chemotherapeutic, for example, an additionalkinase inhibitor, PD-1 inhibitor, or BCL-2 inhibitor, or combinationthereof.

BACKGROUND

The regulation of the cell cycle is governed and controlled by specificproteins, which are activated and deactivated mainly throughphosphorylation/dephosphorylation processes in a precisely timed manner.The key proteins that coordinate the initiation, progression, andcompletion of cell-cycle program are cyclin dependent kinases (CDKs).Cyclin-dependent kinases belong to the serine-threonine protein kinasefamily. They are heterodimeric complexes composed of a catalytic kinasesubunit and a regulatory cyclin subunit. CDK activity is controlled byassociation with their corresponding regulatory subunits (cyclins) andCDK inhibitor proteins (Cip & Kip proteins, INK4s), by theirphosphorylation state, and by ubiquitin-mediated proteolytic degradation(see D. G. Johnson, C. L. Walker, Annu. Rev. Pharmacol. Toxicol 39(1999) 295-312; D. O. Morgan, Annu. Rev. Cell Dev. Biol. 13 (1997)261-291; C. J. Sherr, Science 274 (1996) 1672-1677; T. Shimamura et al.,Bioorg. Med. Chem. Lett. 16 (2006) 3751-3754).

There are four CDKs that are significantly involved in cellularproliferation: CDK1, which predominantly regulates the transition fromG2 to M phase, and CDK2, CDK4, and CDK6, which regulate the transitionfrom G1 to S phase (Malumbres M, Barbacid M. Cell cycle, CDKs andcancer: a changing paradigm. Nat. Rev. Cancer 2009; 9(3):153-166). Inearly to mid G1 phase, when the cell is responsive to mitogenic stimuli,activation of CDK4-cyclin D and CDK6-cyclin D induces phosphorylation ofthe retinoblastoma protein (pRb). Phosphorylation of pRb releases thetranscription factor E2F, which enters the nucleus to activatetranscription of other cyclins which promote further progression of thecell cycle (see J. A. Diehl, Cancer Biol. Ther. 1 (2002) 226-231; C. J.Sherr, Cell 73 (1993) 1059-1065). CDK4 and CDK6 are closely relatedproteins with basically indistinguishable biochemical properties (see M.Malumbres, M. Barbacid, Trends Biochem. Sci. 30 (2005) 630-641).

A number of CDK 4/6 inhibitors have been identified, including specificpyrido[2,3-d]pyrimidines, 2-anilinopyrimidines, diaryl ureas,benzoyl-2,4-diaminothiazoles, indolo[6,7-a]pyrrolo[3,4-c]carbazoles, andoxindoles (see P. S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer DrugTargets 8 (2008) 53-75). For example, WO 03/062236 identifies a seriesof 2-(pyridin-2-ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment ofRb positive cancers that show selectivity for CDK4/6, including6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one(PD0332991), which is currently being tested by Pfizer in late stageclinical trials as an anti-neoplastic agent against estrogen-positive,HER2-negative breast cancer. Tate, et al. describe the antitumoractivity of the CDK4/6 inhibitor abemaciclib (LY2835219)(“Semi-Mechanistic Pharmacokinetic/Pharmacodynamic Modeling of theAntitumor Activity of LY2835219, a New Cyclin-Dependent Kinase 4/6Inhibitor, in Mice Bearing Human Tumor Xenografts”, Clin Cancer Res(Jul. 15, 2014) 20; 3763-74). Rader, et al. describe the reducedproliferation in neuroblastoma-derived cell lines using the CDK4/6inhibitor ribociclib (LEE011) (“Dual CDK4/CDK6 Inhibition Induces CellCycle Arrest and Senescence in Neorbalstoma”, Clin Cancer Res (Nov. 15,2013) 19(22): 6173-82). VanderWel et al. describe an iodine-containingpyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent and selective CDK4inhibitor (see VanderWel et al., J. Med. Chem. 48 (2005) 2371-2387). WO99/15500 filed by Glaxo Group Ltd discloses protein kinase andserine/threonine kinase inhibitors. WO 2010/020675 filed by Novartis AGdescribes pyrrolopyrimidine compounds as CDK inhibitors. WO 2011/101409also filed by Novartis describes pyrrolopyrimidines with CDK 4/6inhibitory activity. WO 2005/052147 filed by Novartis and WO 2006/074985filed by Janssen Pharma disclose additional CDK4 inhibitors. WO2012/061156 filed by Tavares and assigned to G1 Therapeutics describesCDK inhibitors. WO 2013/148748 filed by Francis Tavares and assigned toG1 Therapeutics describes Lactam Kinase Inhibitors. PCT PatentApplication No. PCT/US2014/029073 filed by Strum et al. and assigned toG1 Therapeutics describes compounds and methods for protection ofhematopoietic stem and progenitor cells against ionizing radiation usingCDK4/6 inhibitors. PCT Patent Application No. PCT/US2014/028685 filed byStrum et al. and assigned to G1 Therapeutics describes compounds andmethods for protection of normal cells during chemotherapy using CDK4/6inhibitors. PCT Patent Application No. PCT/US2014/029429 filed by Strumet al. and assigned to G1 Therapeutics describes compounds and methodsfor treating Rb-positive cancers using CDK4/6 inhibitors. PCT PatentApplication No. PCT/US2014/029274 filed by Strum et al. and assigned toG1 Therapeutics describes compounds and methods for treating certaincancers with CDK4/6 inhibitors.

It has recently been reported that the Pfizer CDK4/6 inhibitorpalbociclib in combination with letrozole, while increasing progressionfree survival (PFS) compared with letrozole alone in post-menopausalwomen with estrogen receptor positive (ER+), human epidermal growthfactor receptor 2 negative (HER2−) locally advanced or metastatic breastcancer, failed to statistically extend overall survival (OS), animportant secondary endpoint. Based on the events at the time of theassessment, a median OS of 37.5 months was observed in the combination(paalbociclib+letrozole) arm versus 33.3 months for those who receivedletrozole alone, a difference of 4.2 months (HR=0.813, 95% CI. 0.492,1.345). This OS observation at the time of final PFS analysis was notstatistically significant.

Accordingly, there is an ongoing need for methods, combinations, anddosing regimens to treat patients with select Rb-positive cancers andabnormal cellular proliferative disorders.

SUMMARY OF THE INVENTION

Methods, combinations, and dosing regimens are provided to treat selectRb-positive abnormal cellular proliferation disorders in a hostincluding an Rb-positive cancer using a CDK4/6 inhibitor describedherein.

In one aspect, a method is provided that includes the administering to ahost in need thereof an effective amount of a CDK4/6 inhibitor describedherein in combination or alternation with at least one additionalchemotherapeutic agent. The treatment regimen includes theadministration of a CDK4/6 inhibitor described herein in combination oralternation with at least one additional kinase inhibitor. In oneembodiment, the at least one additional kinase inhibitor is selectedfrom a phosphoinositide 3-kinase (PI3K) inhibitor, a Bruton's tyrosinekinase (BTK) inhibitor, a spleen tyrosine kinase (Syk) inhibitor, aMitogen-activated protein kinase kinase (MEK) inhibitor, a RapidlyAccelerated Fibrosarcoma (Raf) kinase inhibitor, a B-cell lymphoma 2(Bcl-2) protein inhibitor, a programmed death protein 1 (PD-1)inhibitor, or a combination thereof.

Without wanting to be bound by any particular theory, the use of theCDK4/6 inhibitors described herein, in combination with the kinaseinhibitors described herein, provide enhanced additive anticancereffects while believed to reduce disease progression associated withkinase inhibitor resistance that commonly develops in kinase inhibitortherapy which leads to disease progression.

In one embodiment, the CDK4/6 inhibitor is combined in a single dosageform with the at least one additional chemotherapeutic agent.

As contemplated herein, the CDK4/6 inhibitor to be administered isselected from the compounds of Formula I, II, III, IV, or V as describedherein, or a pharmaceutically acceptable composition, salt, isotopicanalog, or prodrug thereof. In one non-limiting example, a CDK4/6inhibitor can be selected from the compounds of Table 1 below, or apharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof. In a further embodiment, the CDK4/6 inhibitor isselected from compounds Q, T, U, or GG, and the chemotherapeutic agentis selected from a phosphoinositide 3-kinase (PI3K) inhibitor, aBruton's tyrosine kinase (BTK) inhibitor, a spleen tyrosine kinase (Syk)inhibitor, a Raf inhibitor, a MEK inhibitor, a programmed death protein1 (PD-1) inhibitor, or a B-cell lymphoma 2 (Bcl-2) protein inhibitor. Inan additional embodiment, the CDK4/6 inhibitor is selected fromcompounds X or BB, and the chemotherapeutic agent is selected from aphosphoinositide 3-kinase (PI3K) inhibitor, a Bruton's tyrosine kinase(BTK) inhibitor, a spleen tyrosine kinase (Syk) inhibitor, a Rafinhibitor, a MEK inhibitor, a programmed death protein 1 (PD-1)inhibitor, or a B-cell lymphoma 2 (Bcl-2) protein inhibitor.

In a specific embodiment, a CDK4/6 inhibitor selected from the compoundsof Formula I, II, III, IV, or V as described herein, or apharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof, is administered in combination or alternation with thePI3 kinase inhibitor pictilisib (GDC-0941; Genentech Inc., South SanFrancisco, Calif.) to a host suffering from colorectal, breast, softtissue sarcoma, melanoma, ovarian, gastric, or prostate cancer. In aspecific embodiment, the CDK4/6 inhibitor is selected from compound Q,T, U, GG, X, or BB. In a more particular embodiment, the CDK4/6inhibitor is compound GG and the host is suffering from breast cancer.

As further contemplated herein, the CDK4/6 inhibitor is combined oralternated with another chemotherapeutic agent to treat alymphohematopoietic malignancy, for example, but not limited to, B-celllineage leukemia or lymphoma, T-cell lineage leukemia or lymphoma, ormyeloid-lineage leukemia or lymphoma, for example, but not limited toacute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). Inone embodiment, the CDK4/6 inhibitor is selected from Compound Q, T, U,or GG and the at least one additional chemotherapeutic agent is a kinaseinhibitor. In one embodiment, the CDK4/6 inhibitor is selected fromCompound X or BB and the at least one additional chemotherapeutic agentis a kinase inhibitor. In one embodiment, the kinase inhibitor is a BTKinhibitor or a Syk inhibitor. In one embodiment, the BTK inhibitor isibrutinib. In one embodiment, the BTK inhibitor is ACP-196.

In one aspect contemplated herein, the CDK4/6 inhibitor is combined oralternated with another chemotherapeutic agent to treat a solid tumor.In one embodiment, the solid tumor is Erb-2/human epidermal growthfactor receptor (HER)-2-positive breast cancer. In one embodiment, thesolid tumor is a non-small cell lung cancer. In one embodiment, thesolid tumor is a colorectal cancer. In one embodiment, the CDK4/6inhibitor is selected from Compound Q, T, U, or GG and the at least oneadditional chemotherapeutic agent is a kinase inhibitor. In oneembodiment, the CDK4/6 inhibitor is selected from Compound X or BB andthe at least one additional chemotherapeutic agent is a kinaseinhibitor. In one embodiment, the kinase inhibitor is a BTK inhibitor ora Syk inhibitor. In one embodiment, the BTK inhibitor is ibrutinib. Inone embodiment, the BTK inhibitor is ACP-196.

In one aspect, a method is provided for the treatment of an Rb-positivecancer comprising administering an effective amount of a CDK 4/6inhibitor compound of Formula I, II, III, IV, or V in combination with aBruton's tyrosine kinase (BTK) inhibitor to a host in need thereof,wherein the Bruton's tyrosine kinase (BTK) inhibitor is selected fromibrutinib or ACP-196.

In another aspect, a method is provided for the treatment of anRb-positive cancer comprising administering an effective amount of a CDK4/6 inhibitor compound of Formula I, II, III, IV, or V in combinationwith a spleen tyrosine kinase (SYK) inhibitor.

In an additional aspect, a method is provided for the treatment of anRb-positive cancer comprising administering an effective amount of a CDK4/6 inhibitor compound of Formula I, II, III, IV, or V in combinationwith a PI3 kinase inhibitor to a host in need thereof, wherein the PI3kinase inhibitor is pictilisib.

In one aspect, a method is provided for the treatment of an Rb-positivecancer in a host comprising: a) administering to the host having anRb-positive cancer an effective amount of a CDK 4/6 inhibitor compoundof the Formula I, II, III, IV, or V, wherein the compound is capable ofarresting a significant portion of the Rb-positive abnormal cells in theG0 or G1 phase of the cell cycle; b) administering to the host at leastone additional chemotherapeutic agent within about 48 hours ofadministration of the CDK 4/6 inhibitor.

In one aspect, a method is provided comprising 1) administering to ahost having an Rb-positive abnormal cellular proliferation disorder aCDK4/6 inhibitor described herein, wherein the CDK4/6 inhibitor iscapable of arresting a significant portion of the Rb-positive abnormalcells in the G0 or G1 phase of the cell cycle, 2) administering to thehost at least one additional chemotherapeutic agent just prior to, or inone alternative concomitantly to, a significant portion of theRb-positive abnormal cells, for example at least 50%, at least 60%, atleast 70%, at least 80%, reentering the cell cycle, for example, withinabout 12 hours, within about 14 hours, within about 16 hours, withinabout 18 hours, within about 20 hours, within about 24 hours, withinabout 30 hours, within about 36 hours, within about 40 hours, or withinabout 48 hours of administration of the CDK 4/6 inhibitor, wherein, dueto the synchronous reentry of the abnormal cells into the cell cycle,the efficacy of the chemotherapeutic agent is enhanced. Accordingly, theCDK4/6 inhibitors described herein can be combined or alternated withanother chemotherapeutic described herein to treat a select Rb-positiveabnormal cellular proliferation disorder, such as a cancer, in order tosynchronize the Rb-positive abnormal cells so that more abnormal cellsare exposed to the chemotherapeutic agent at the cell-cycle stage thatthe chemotherapeutic agent is most effective, e.g., G1 phase, S phase,G2 phase, or M phase. Specifically, the invention includes administeringto a patient having an Rb-positive abnormal cellular proliferationdisorder, such as cancer, an effective amount of a CDK4/6 inhibitordescribed herein, wherein the compound has a pharmacokinetic andenzymatic half-life that provides for a transient, reversible G1 arrestof the Rb-positive abnormal cells, allowing for an initial halt of asignificant portion of the cells in the G0 or G1 phase of the cellcycle, followed by the synchronous reentry of these cells into the cellcycle, wherein the administration of at least one additionalchemotherapeutic agent is timed so that the abnormal cells are enteringa cell-cycle stage that the chemotherapeutic agent is most effective.The chemotherapeutic agent can be any of those described in thisapplication or utilized in the standards of care used to treat abnormalcellular proliferation. In one embodiment, the chemotherapeutic agent isa kinase inhibitor.

Non-limiting examples of CDK4/6 inhibitors useful in the presentinvention are described in Table 1, or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof as providedbelow. In one embodiment, the solid tumor is a colorectal cancer. In oneembodiment, the CDK4/6 inhibitor is selected from Compound Q, T, U, orGG and the at least one additional chemotherapeutic agent is a kinaseinhibitor. In one embodiment, the CDK4/6 inhibitor is selected fromCompound X or BB and the at least one additional chemotherapeutic agentis a kinase inhibitor.

Thus in one embodiment, the invention includes administering a CDK4/6inhibitor described herein, for example a CDK4/6 inhibitor selected fromTable 1, in an effective amount to treat a host suffering from anRb-positive abnormal cellular proliferation disorder in a treatmentregimen, wherein (either alone or in any combination thereof, each ofwhich is considered specifically and independently described): (i) asubstantial portion of the abnormal cells (e.g., at least 50%, at least60%, at least 70%, at least 80% or greater) are arrested in G0 or G1 andre-enter the cell cycle in less than about 24 hours, 30 hours, 36 hours,or 48 hours from the last administration of a CDK4/6 inhibitor describedherein; (ii) a substantial portion of the abnormal cells reenter thecell-cycle synchronously in less than about 24 hours, 30 hours, 36hours, or 48 hours from the last administration of the CDK4/6 inhibitordescribed herein; (iii) the dissipation of the CDK4/6 inhibitor'sinhibitory effect on the abnormal cells occurs in less than about 24hours, 30 hours, 36 hours, or 48 hours from the administration of theCDK4/6 inhibitor; (iv) a substantial portion of the abnormal cellsreenter the cell-cycle in less than about 24 hours, 30 hours, 36 hours,or 48 hours from the dissipation of the CDK4/6 inhibitor's inhibitoryeffect; or (vi) a substantial portion of the abnormal cells reenter thecell-cycle within less than about 24 hours, about 30 hours, about 36hours, or about 48 hours from the point in which the administered CDK4/6inhibitor compound's concentration level in the subject's blood dropsbelow a therapeutic effective concentration, and, either just prior toor concomitantly with the re-entry of the abnormal cells as described in(i)-(vi), administering at least one additional chemotherapeutic agent.

In a central embodiment of the invention, a compound described hereincan be administered in a concerted regimen with another agent such as aDNA- or non-DNA-damaging, anti-neoplastic agent, for example asdescribed below, for beneficial, additive, or synergistic effect againstRB-positive abnormal cellular proliferation. By combining theadministration of a CDK4/6 inhibitor described herein with the timelyadministration of additional chemotherapeutic agents, for example, atthe time point wherein a larger number of abnormal cells are mostsusceptible to the at least one additional chemotherapeutic agent'sdeleterious effects due to synchronous re-entry into the cell cyclecaused by administration of the CDK4/6 inhibitor, it is possible for thehealth care practitioner to decrease the amount of the additionalchemotherapeutic agent to minimize the unwanted adverse effects whileachieving an increased efficacy of the desired therapeutic benefit.

In certain embodiments, a CDK4/6 inhibitor described herein isadministered to the host prior to treatment with anotherchemotherapeutic agent, during treatment with another chemotherapeuticagent, after administration of another chemotherapeutic agent, or acombination thereof. In one embodiment, a CDK4/6 inhibitor describedherein is administered to the subject less than about 48 hours, 40hours, 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, or 4hours or less prior to treatment with the other chemotherapeutic agentin order to sensitize the Rb-positive cancer to the chemotherapeuticagent. In one embodiment, a CDK4/6 inhibitor described herein isadministered up to 48 hours prior to treatment with the otherchemotherapeutic agent.

In another aspect of the present invention, provided herein is a methodfor treating T-cell malignancies using CDK4/6 inhibitors describedherein. In one embodiment of the present invention, provided herein is amethod of treating a host suffering from a T-cell malignancy comprisinga) administering to the host a CDK4/6 inhibitor of the Formula I, II,III, IV, or V, b) analyzing the cell cycle status of T-cell malignantcells and at least one subset of non-diseased hematopoetic cells, c)adjusting the dose of the CDK4/6 inhibitor so that a significant portionof the T-cell malignant cells are in G0 or G1 cell cycle arrest and atleast one subset of non-diseased hematopoietic cells are not in G0 or G1arrest.

In one embodiment of the present invention, provided herein is a methodof treating a host suffering from a T-cell malignancy comprising, a)administering to the host a CDK4/6 inhibitor of the Formula I, II, III,IV, or V, b) analyzing the cell cycle status of T-cell malignant cellsand at least one subset of non-diseased hematopoetic cells, c) adjustingthe dose of the CDK4/6 inhibitor so that a significant portion of theT-cell malignant cells are not in G0 or G1 arrest and at least onesubset of non-diseased hematopoietic cells are in G0 or G1 arrest, d)administering a chemotherapeutic agent.

In one aspect of the invention, provided herein is a method of treatingan Rb-positive T-cell malignancy in a host by administering a CDK4/6inhibitor described herein at a dose that provides for the long terminhibition of the proliferation of the T-cell malignancy, but providesfor a differential inhibition of non-diseased Rb-positive hematopoieticcells, for example, hematopoietic stem cells (HSCs), multipotentprogenitors (MPPs), hematopoietic progenitor cells (HPCs), and otherhematological cells, which allows these non-diseased hematopoietic cellsto reenter the cell-cycle sooner than the diseased T-cell, thus allowingfor the prolonged use of these CDK4/6 inhibitors to treat the T-cellmalignancy while providing for a reduction in the side-effectsassociated with the use of CDK4/6 inhibitors generally. In oneembodiment of the invention, provided herein is a method of treating ahost suffering from a T-cell malignancy comprising administering to thehost a CDK4/6 inhibitor of the Formula I, II, III, IV, or V, measuringor analyzing the cell cycle status of T-cell malignant cells andnon-diseased hematopoetic cells, for example but not limited to, longterm hematopoietic stem cells (LT-HSCs), short term hematopoietic stemcells (ST-HSCs), hematopoietic progenitor cells (HPCs), multipotentprogenitors (MPPs), oligodendrocyte pre-progenitors (OPPs), monocyteprogenitors, granulocyte progenitors, common myeloid progenitors (CMPs),common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors(GMPs), granulocyte progenitors, monocyte progenitors, andmegakaryocyte-erythroid progenitors (MEPs), megakaryocyte progenitors,erythroid progenitors, or a combination thereof, adjusting the dose ofthe CDK4/6 inhibitor so that a significant portion of the T-cellmalignant cells are in G0 or G1 cell cycle arrest and at least onesubset of non-diseased hematopoietic cells are not in G0 or G1 arrest.In a particular embodiment, the host is a human, and the non-diseasedhematological lineage cells are selected from HSC/MPPs(CD45dim/CD34+/CD38−), OPPs (CD45dim/CD34+/CD38+), monocyte progenitors(CD45+/CD14+/CD11b+), granulocyte progenitors (CD45+/CD14−/CD11b+),erythroid progenitors (CD45−/CD71+), and megakaryocyte progenitors(CD45+/CD61+).

In an alternative embodiment, a CDK 4/6 inhibitor can be administered inan amount so that a significant portion, for example at least 50%, atleast 60%, at least 70%, at least 80%, of the diseased T-cells are inactive cell cycling while a significant portion of at least one subsetof non-diseased hematopoietic cell lineage cells, due to the effects ofthe CDK 4/6 inhibitor, are arrested in the G0 and/or G1 phase of thecell cycle, and subsequently administering a second chemotherapeuticagent.

It has been discovered that hematological cells respond to CDK4/6inhibitors in a differential fashion. Accordingly, this differentialresponse provides for the differential dosing of a CDK4/6 inhibitorbased on the specific hematological disorder the host may be sufferingfrom. Thus, in one aspect of the invention, provided herein is a methodof treating a host suffering from a hematological cellular proliferationdisorder comprising 1) identifying the specific hematologicaldeficiency, 2) administering to the host a CDK4/6 inhibitor in an amountsufficient to inhibit the proliferation of the specific hematologicaldeficiency, 3) analyzing the cell cycle status of the specific diseasedhematological cells and one or more non-diseased hematological celllineages 4) adjusting the dose of the CDK4/6 inhibitor to the minimalamount necessary so that the diseased cells remain inhibited, whileallowing at least one subset of non-diseased hematological cells toproliferate. As contemplated herein, the non-diseased hematopoetic cellscan be, for example, but not limited to, long term hematopoietic stemcells (LT-HSCs), short term hematopoietic stem cells (ST-HSCs),hematopoietic progenitor cells (HPCs), multipotent progenitors (MPPs),oligodendrocyte pre-progenitors (OPPs), monocyte progenitors,granulocyte progenitors, common myeloid progenitors (CMPs), commonlymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs),granulocyte progenitors, monocyte progenitors, andmegakaryocyte-erythroid progenitors (MEPs), megakaryocyte progenitors,erythroid progenitors, or a combination thereof, adjusting the dose ofthe CDK4/6 inhibitor so that a significant portion of the T-cellmalignant cells are in G0 or G1 cell cycle arrest and at least onesubset of non-diseased hematopoietic cells are not in G0 or G1 arrest.In a particular embodiment, the host is a human, and the non-diseasedhematological lineage cells are selected from HSC/MPPs(CD45dim/CD34+/CD38−), OPPs (CD45dim/CD34+/CD38+), monocyte progenitors(CD45+/CD14+/CD11b+), granulocyte progenitors (CD45+/CD14−/CD11b+),erythroid progenitors (CD45−/CD71+), and megakaryocyte progenitors(CD45+/CD61+).

In one aspect of the present invention, the differential effect theCDK4/6 inhibitors described herein have on hematological cells at thesame dosage provides for a method of protecting certain cell linagesduring chemotherapeutic treatment using a DNA-damaging agent to treat aproliferative disorder, for example, an Rb-positive cancer, wherein theCDK4/6 inhibitor is used to protect certain hematopoetic cell lineages,and wherein the dosing of the CDK4/6 inhibitor can be differentiallyadjusted based on the effect the chemotherapeutic agent has on aparticular hematological cell line. Thus, for example, if a certainhematological cell line is adversely effected by the chemotherapeutic,the CDK4/6 inhibitor dose can be adjusted to allow the particularlineage to replicate while still affording some chemoprotection to otherhematopoetic cells. As contemplated herein, the non-diseasedhematopoetic cells can be, for example, but not limited to, long termhematopoietic stem cells (LT-HSCs), short term hematopoietic stem cells(ST-HSCs), hematopoietic progenitor cells (HPCs), multipotentprogenitors (MPPs), oligodendrocyte pre-progenitors (OPPs), monocyteprogenitors, granulocyte progenitors, common myeloid progenitors (CMPs),common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors(GMPs), granulocyte progenitors, monocyte progenitors, andmegakaryocyte-erythroid progenitors (MEPs), megakaryocyte progenitors,erythroid progenitors, or a combination thereof, adjusting the dose ofthe CDK4/6 inhibitor so that a significant portion of the T-cellmalignant cells are in G0 or G1 cell cycle arrest and at least onesubset of non-diseased hematopoietic cells are not in G0 or G1 arrest.In a particular embodiment, the host is a human, and the non-diseasedhematological lineage cells are selected from HSC/MPPs(CD45dim/CD34+/CD38−), OPPs (CD45dim/CD34+/CD38+), monocyte progenitors(CD45+/CD14+/CD11b+), granulocyte progenitors (CD45+/CD14−/CD11b+),erythroid progenitors (CD45−/CD71+), and megakaryocyte progenitors(CD45+/CD61+).

In some embodiments, the subject or host is a mammal, including a human.The compound can be administered to the subject by any desired route,including intravenous, sublingual, buccal, oral, intraaortal, topical,intranasal, parenteral, transdermal, systemic, intramuscular, or viainhalation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of the percentage of cells in the G0-G1 phase of thecell cycle vs. time after washout of the compound (hours) in humanfibroblast (Rb-positive) cells. FIG. 1B is a graph of the percentage ofcells in the S phase of the cell cycle vs. time after washout of thecompound (hours) in human fibroblast (Rb-positive) cells. FIG. 1C is agraph of the percentage of cells in the G0-G1 phase of the cell cyclevs. time after washout of the compound (hours) in human renal proximaltubule epithelial (Rb-positive) cells. FIG. 1D is a graph of thepercentage of cells in the S phase of the cell cycle vs. time afterwashout of the compound (hours) in human renal proximal tubuleepithelial (Rb-positive) cells. These cellular wash out experimentsdemonstrated that the inhibitor compounds of the present invention havea short, transient G1-arresting effect in different cell types. Theeffect on the cell cycle following washing out of the compounds wasdetermined at 24, 36, 40, and 48 hours. As described in Example 153, theresults show that cells treated with

FIGS. 2-4 illustrate several exemplary embodiments of R² of thecompounds useful in the present invention.

FIGS. 5A-5C, 6A-6D, 7A-7C, 8A-8B, and 9A-9F illustrate several exemplaryembodiments of the core structure of the compounds useful in the presentinvention.

FIG. 10A is a graph of the percentage of cells in G2-M phase (opencircles), S phase (triangles), G0-G1 phase (squares), <2N (diamonds) vs.variable concentration (nM) of compound T in tHS68 cells. TheCDK4/6-dependent cell line (tHS68)(Rb-positive) was treated with theindicated concentrations of Compound T for 24 hours. Following treatmentof Compound T, cells were harvested and analyzed for cell cycledistribution. As described in Example 154, tHS68 cells show a clean G1arrest accompanied by a corresponding decrease in the number of cells inS-phase.

FIG. 10B is a graph of the number of tHS68 cells (Rb-positive) vs. theDNA content of the cells (as measured by propidium iodide). Cells weretreated with DMSO for 24 hours, harvested, and analyzed for cell cycledistribution.

FIG. 10C is a graph of the number of WM2664 cells (Rb-positive) vs. theDNA content of the cells (as measured by propidium iodide). Cells weretreated with DMSO for 24 hours, harvested, and analyzed for cell cycledistribution.

FIG. 10D is a graph of the number of A2058 cells (CDK4/6-independentcell line) vs. the DNA content of the cells (as measured by propidiumiodide). Cells were treated with DMSO for 24 hours, harvested, andanalyzed for cell cycle distribution.

FIG. 10E is a graph of the number of tHS68 cells (Rb-positive) vs. theDNA content of the cells (as measured by propidium iodide) aftertreatment with Compound T. Cells were treated with Compound T (300 nM)for 24 hours, harvested, and analyzed for cell cycle distribution. Asdescribed in Example 154, treatment of tHS68 cells with Compound Tcauses a loss of the S-phase peak (indicated by arrow).

FIG. 10F is a graph of the number of WM2664 cells (Rb-positive) vs. theDNA content of the cells (as measured by propidium iodide) aftertreatment with Compound T. Cells were treated with Compound T (300 nM)for 24 hours, harvested, and analyzed for cell cycle distribution. Asdescribed in Example 154, treatment of WM2664 cells with Compound Tcauses a loss of the S-phase peak (indicated by arrow).

FIG. 10G is a graph of the number of A2058 cells (Rb-negative) vs. theDNA content of the cells (as measured by propidium iodide) aftertreatment with Compound T. Cells were treated with Compound T (300 nM)for 24 hours, harvested, and analyzed for cell cycle distribution. Asdescribed in Example 154, treatment of A2058 cells with Compound T doesnot cause a loss of the S-phase peak (indicated by arrow).

FIG. 11 is a Western blot showing the phosphorylation levels of Rb atSer807/811 and Ser780 after treatment with Compound T. Rb-positive(tHS68 or WM2664) and Rb-negative cell lines (A2058) were treated withCompound T (300 nM) for the indicated times (0, 4, 8, 16, and 24 hours).MAPK levels are shown as a control for protein levels. Followingtreatment, cells were harvested and analyzed for Rb-phosphorylation bywestern blot analysis. As described in Example 155, Compound T treatmentresulted in reduced Rb-phosphorylation starting 16 hours after treatmentin CDK4/6-dependent cell lines (tHS68 and WM2664), but not in theCDK4/6-independent cell line (A2058).

FIG. 12 is a graph of 5′-ethynyl-2′-deoxyuridine (EdU) incorporation vs.time after administration (minutes) of Compound T to healthy C57BL/6female mice. Compound T (50 mg/kg or 100 mg/kg) was administered by i.p.injection to assess the temporal effect of transient CDK4/6 inhibitionon bone marrow arrest. As described in Example 156, a single i.p. doseof Compound T results in a dose and time dependent reduction andrecovery of proliferating cells.

FIG. 13 is a graph of EdU incorporation into thymocytes vs. time afteradministration (minutes) of Compound T to healthy C57BL/6 female mice(circles; 50 mg/kg), (squares; 100 mg/kg).

FIG. 14 is a graph illustrating Mac1+/Gr1+ cells and EdU incorporationvs. time after administration (minutes) of Compound T to healthy C57BL/6female mice (circles; 50 mg/kg), (squares; 100 mg/kg). Mac1+/Gr1+ is amarker of of myeloid cells.

FIG. 15 is a graph illustrating B220+ cells and EdU incorporation vs.time after administration (minutes) of Compound T to healthy C57BL/6female mice (circles; 50 mg/kg), (squares; 100 mg/kg). B220+ is a markerof B lymphoid cells.

FIG. 16 is a graph illustrating Ter119+ cells and EdU incorporation vs.time after administration (minutes) of Compound T to healthy C57BL/6female mice (circles; 50 mg/kg), (squares; 100 mg/kg). Ter119+ is amarker of erythroid cells.

FIG. 17 is a graph illustrating LK+ cells and EdU incorporation vs. timeafter administration (minutes) of Compound T to healthy C57BL/6 femalemice (circles; 50 mg/kg), (squares; 100 mg/kg). LK+(LK (Lin−Sca-1−C-kit+) is a marker of myeloid progenitor cells.

FIG. 18 is a graph illustrating LSK+ cells and EdU incorporation vs.time after administration (minutes) of Compound T to healthy C57BL/6female mice (circles; 50 mg/kg), (squares; 100 mg/kg). LSK+(Lin−Sca-1+c-kit+) is a marker of hematopoietic stem cells.

FIG. 19 is a graph of EdU incorporation into hematopoietic stem cells(HSCs) vs. time after administration (minutes) of Compound T to healthyC57BL/6 female mice. Compound T (50 mg/kg or 100 mg/kg) was administeredby i.p. injection.

FIG. 20 is a graph showing tumor volume (cubic mm) vs. time afterinitiation of treatment (days) in a mouse model of breast cancer.Immunodeficient mice were implanted with the human breast cancer cellline MCF7 (an Rb-positive cell line). Once the tumors reached sufficientsize, the mice were randomized into treatment cohorts. Mice were treatedwith vehicle control (small circles), 100 mg/kg Compound GG (days 1-28)(small squares), 100 mg/kg palbociclib (days 1-28) (large squares), 50mg/kg Compound GG (days 1-28) and 100 mg/kg GDC-0941 (days 15-28)(triangles), 10 mg/kg Compound GG (days 1-28) and 100 mg/kg GDC-0941(days 15-28) (upside down triangles), 100 mg/kg GDC-0941 (days 1-28)(diamonds), or 100 mg/kg Compound GG (days 1-28) and 100 mg/kg GDC-0941(days 1-28) (large circles). As discussed in Example 157, mice treatedwith a combination of 100 mg/kg Compound GG and 100 mg/kg GDC-0941 for28 days showed enhanced efficacy toward the MCF7 breast cancer cell lineimplants, as compared to mice treated with vehicle control, 100 mg/kgCompound GG, 100 mg/kg palbociclib, 50 mg/kg Compound GG and 100 mg/kgGDC-0941, 10 mg/kg Compound GG and 100 mg/kg GDC-0941, or 100 mg/kgGDC-0941.

FIG. 21A is a graph showing white blood cell counts (cells/μl) at Day 14in mice (human breast cancer cell line MCF7 implant model) treated withvehicle only (circles), 10 mg/kg Compound GG (squares), 50 mg/kgCompound GG (triangles), or 100 mg/kg Compound GG (upside downtriangles). See Example 157.

FIG. 21B is a graph showing lymphocyte counts (cells/μl) at Day 14 inmice (human breast cancer cell line MCF7 implant model) treated withvehicle only (circles), 10 mg/kg Compound GG (squares), 50 mg/kgCompound GG (triangles), or 100 mg/kg Compound GG (upside downtriangles). See Example 157.

FIG. 21C is a graph showing neutrophil counts (cells/μl) at Day 14 inmice (human breast cancer cell line MCF7 implant model) treated withvehicle only (circles), 10 mg/kg Compound GG (squares), 50 mg/kgCompound GG (triangles), or 100 mg/kg Compound GG (upside downtriangles). See Example 157.

FIG. 21D is a graph showing monocyte counts (cells/μl) at Day 14 in mice(human breast cancer cell line MCF7 implant model) treated with vehicleonly (circles), 10 mg/kg Compound GG (squares), 50 mg/kg Compound GG(triangles), or 100 mg/kg Compound GG (upside down triangles). SeeExample 157.

FIG. 21E is a graph showing platelet counts (cells/μl) at Day 14 in mice(human breast cancer cell line MCF7 implant model) treated with vehicleonly (circles), 10 mg/kg Compound GG (squares), 50 mg/kg Compound GG(triangles), or 100 mg/kg Compound GG (upside down triangles). SeeExample 157.

FIG. 21F is a graph showing red blood cell counts (cells/μl) at Day 14in mice (human breast cancer cell line MCF7 implant model) treated withvehicle only (circles), 10 mg/kg Compound GG (squares), 50 mg/kgCompound GG (triangles), or 100 mg/kg Compound GG (upside downtriangles). See Example 157.

DETAILED DESCRIPTION OF THE INVENTION

Methods, combinations, and dosing regimens are provided to treat selectRb-positive abnormal cellular proliferation disorders in a hostincluding an Rb-positive cancer using a CDK4/6 inhibitor describedherein in combination with at least one additional chemotherapeuticagent, for example, a specific kinase inhibitor. In an alternativeaspect, the CDK4/6 inhibitors described herein are used to induce aG0-G1 cell-cycle arrest in Rb-positive tumors in order to synchronizethe tumor cell upon dissipation of the inhibitory CDK4/6 effect,resulting in an increased exposure by a larger number of cells during asecond chemotherapeutic's most efficacious period, e.g., G1, S, G2, or Mphase. In a further aspect, provided herein is a method of treating anRb-positive T-cell malignancy by administering a CDK4/6 inhibitorcompound described herein at a dose that provides for an extended periodof the inhibition of the growth of the T-cell malignancy, but providesfor a quicker reentry into the cell-cycle of non-diseased Rb-positivehematopoietic cells, for example, hematopoietic stem and progenitorcells (HSPCs). In a further aspect, provided herein is a method fordifferentially protecting hematopoetic cell lineages during treatment ofa cancer using a DNA-damaging agent based on the particular dose ortiming of the dose of the CDK4/6 inhibitor administered to the host.

Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. As used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Definition of standard chemistryterms may be found in reference works, including Carey and Sundberg(2007) Advanced Organic Chemistry 5^(th) Ed. Vols. A and B, SpringerScience+Business Media LLC, New York. The practice of the presentinvention will employ, unless otherwise indicated, conventional methodsof synthetic organic chemistry, mass spectroscopy, preparative andanalytical methods of chromatography, protein chemistry, biochemistry,recombinant DNA techniques and pharmacology. Conventional methods oforganic chemistry include those included in March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, 6th Edition, M. B.Smith and J. March, John Wiley & Sons, Inc., Hoboken, N.J., 2007.

The term “alkyl,” either alone or within other terms such as “haloalkyl”and “alkylamino,” embraces linear or branched radicals having one toabout twelve carbon atoms. “Lower alkyl” radicals have one to about sixcarbon atoms. Examples of such radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,hexyl and the like. The term “alkylene” embraces bridging divalentlinear and branched alkyl radicals. Examples include methylene,ethylene, propylene, isopropylene and the like.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about twelve carbon atoms.“Lower alkenyl” radicals having two to about six carbon atoms. Examplesof alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyland 4-methylbutenyl. The terms “alkenyl” and “lower alkenyl,” embraceradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations.

The term “alkynyl” denotes linear or branched radicals having at leastone carbon-carbon triple bond and having two to about twelve carbonatoms. “Lower alkynyl” radicals having two to about six carbon atoms.Examples of such radicals include propargyl, butynyl, and the like.

Alkyl, alkenyl, and alkynyl radicals may be optionally substituted withone or more functional groups such as halo, hydroxy, nitro, amino,cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.

The term “alkylamino” embraces “N-alkylamino” and “N,N-dialkylamino”where amino groups are independently substituted with one alkyl radicaland with two alkyl radicals, respectively. “Lower alkylamino” radicalshave one or two alkyl radicals of one to six carbon atoms attached to anitrogen atom. Suitable alkylamino radicals may be mono or dialkylaminosuch as N-methylamino, N-ethylamino, N.N-dimethylamino, N,N-diethylaminoand the like.

The term “halo” means halogens such as fluorine, chlorine, bromine oriodine atoms.

The term “haloalkyl” embraces radicals wherein any one or more of thealkyl carbon atoms is substituted with one or more halo as definedabove. Examples include monohaloalkyl, dihaloalkyl and polyhaloalkylradicals including perhaloalkyl. A monohaloalkyl radical, for oneexample, may have an iodo, bromo, chloro or fluoro atom within theradical. Dihalo and polyhaloalkyl radicals may have two or more of thesame halo atoms or a combination of different halo radicals. “Lowerhaloalkyl” embraces radicals having 1-6 carbon atoms. Examples ofhaloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Perfluoroalkyl” means an alkyl radical having allhydrogen atoms replaced with fluoro atoms. Examples includetrifluoromethyl and pentafluoroethyl.

The term “aryl”, alone or in combination, means a carbocyclic aromaticsystem containing one or two rings wherein such rings may be attachedtogether in a fused manner. The term “aryl” embraces aromatic radicalssuch as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. Morepreferred aryl is phenyl. Said “aryl” group may have 1 or moresubstituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro,cyano, alkoxy, lower alkylamino, and the like. An aryl group may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “heterocyclyl” (or “heterocyclo”) embraces saturated, andpartially saturated heteroatom-containing ring radicals, where theheteroatoms may be selected from nitrogen, sulfur and oxygen. In oneembodiment, heterocyclic rings comprise monocyclic 6-8 membered rings,as well as 5-16 membered bicyclic ring systems (which can includebridged fused and spiro-fused bicyclic ring systems). It does notinclude rings containing —O—O—·—O—S— or —S—S— portions. Said“heterocyclyl” group may have one or more substituents such as hydroxyl,Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, loweralkoxy, amino, lower alkylamino, and the like. In one embodiment, said“heterocyclyl” group may have 1 to 3 substituents such as hydroxyl, Boc,halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy,amino, lower alkylamino, and the like. In an alternate embodiment, aheterocyclic ring comprises a monocyclic 3-6 membered ring.

Examples of saturated heterocyclo groups include saturated 3- to6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms[e.g. pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,piperazinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.morpholinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.,thiazolidinyl]. Examples of partially saturated heterocyclyl radicalsinclude dihydrothienyl, dihydropyranyl, dihydrofuryl, dihydrothiazolyl,and the like.

Particular examples of partially saturated and saturated heterocyclogroups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl,thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl,indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl,isochromanyl, chromanyl, 1,2-dihydroquinolyl,1,2,3,4-tetrahydro-isoquinolyl, 1,2,3,4-tetrahydro-quinolyl,2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl,5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl,3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl,2,3-dihydro-1H-lλ′-benzo[d]isothiazol-6-yl, dihydropyranyl, dihydrofuryland dihydrothiazolyl, and the like.

Heterocyclo groups also includes radicals where heterocyclic radicalsare fused/condensed with aryl radicals: unsaturated condensedheterocyclic group containing 1 to 5 nitrogen atoms, for example,indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl,indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g., tetrazolo[1,5-b]pyridazinyl]; unsaturated condensed heterocyclic group containing1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. benzoxazolyl,benzoxadiazolyl]; unsaturated condensed heterocyclic group containing 1to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., benzothiazolyl,benzothiadiazolyl]; and saturated, partially unsaturated and unsaturatedcondensed heterocyclic group containing 1 to 2 oxygen or sulfur atoms[e.g. benzofuryl, benzothienyl, 2,3-dihydro-benzo[1,4]dioxinyl anddihydrobenzofuryl].

The term “heteroaryl” denotes aryl ring systems that contain one or moreheteroatoms selected from the group O, N and S, wherein the ringnitrogen and sulfur atom(s) are optionally oxidized, and nitrogenatom(s) are optionally quarternized. Examples include unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, forexample, pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g.,4H-1,2,4-triazolyl, IH-1,2,3-triazolyl, 2H-1,2,3-triazolyl]; unsaturated5- to 6-membered heteromonocyclic group containing an oxygen atom, forexample, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-memberedheteromonocyclic group containing a sulfur atom, for example, 2-thienyl,3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example,oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl]; unsaturated 5 to 6-memberedheteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g.,1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl].

The term “heteroarylalkyl” denotes alkyl radicals substituted with aheteroaryl group. Examples include pyridylmethyl and thienylethyl.

The term “sulfonyl”, whether used alone or linked to other terms such asalkylsulfonyl, denotes respectively divalent radicals —SO₂—.

The terms “carboxy” or “carboxyl”, whether used alone or with otherterms, such as “carboxyalkyl”, denotes —C(O)—OH.

The term “carbonyl”, whether used alone or with other terms, such as“aminocarbonyl”, denotes —C(O)—.

The term “aminocarbonyl” denotes an amide group of the Formula—C(O)—NH₂.

The terms “heterocycloalkyl” embrace heterocyclic-substituted alkylradicals. Examples include piperidylmethyl and morpholinylethyl.

The term “arylalkyl” embraces aryl-substituted alkyl radicals. Examplesinclude benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkylmay be additionally substituted with halo, alkyl, alkoxy, halkoalkyl andhaloalkoxy.

The term “cycloalkyl” includes saturated carbocyclic groups of 3 to 10carbons. Lower cycloalkyl groups include C₃-C₆ rings. Examples includecyclopentyl, cyclopropyl, and cyclohexyl. Cycloalkyl groups may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “cycloalkylalkyl” embraces cycloalkyl-substituted alkylradicals. “Lower cycloalkylalkyl” radicals are cycloalkyl radicalsattached to alkyl radicals having one to six carbon atoms. Examples ofinclude cyclohexylmethyl. The cycloalkyl in said radicals may beadditionally substituted with halo, alkyl, alkoxy and hydroxy.

The term “cycloalkenyl” includes carbocyclic groups having one or morecarbon-carbon double bonds including “cycloalkyldienyl” compounds.Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl andcycloheptadienyl.

The term “comprising” is meant to be open ended, including the indicatedcomponent but not excluding other elements.

The term “oxo” as used herein contemplates an oxygen atom attached witha double bond.

The term “nitro” as used herein contemplates —NO₂.

The term “cyano” as used herein contemplates —CN.

As used herein, the term “prodrug” means a compound which whenadministered to a host in vivo is converted into the parent drug. Asused herein, the term “parent drug” means any of the presently describedchemical compounds that are useful to treat any of the disordersdescribed herein, or to control or improve the underlying cause orsymptoms associated with any physiological or pathological disorderdescribed herein in a host, typically a human. Prodrugs can be used toachieve any desired effect, including to enhance properties of theparent drug or to improve the pharmaceutic or pharmacokinetic propertiesof the parent. Prodrug strategies exist which provide choices inmodulating the conditions for in vivo generation of the parent drug, allof which are deemed included herein. Nonlimiting examples of prodrugstrategies include covalent attachment of removable groups, or removableportions of groups, for example, but not limited to acylation,phosphorylation, phosphonylation, phosphoramidate derivatives,amidation, reduction, oxidation, esterification, alkylation, othercarboxy derivatives, sulfoxy or sulfone derivatives, carbonylation oranhydride, among others.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist, unless otherwise noted.

The current invention is directed to combinations and methods usingCDK4/6 inhibitors and at least one additional chemotherapeutic agent foruse in the treatment of Rb-positive proliferation disorders.

The term “selective CDK4/6 inhibitor” used in the context of thecompounds described herein includes compounds that inhibit CDK4activity, CDK6 activity, or both CDK4 and CDK6 activity at an IC₅₀ molarconcentration at least about 500 times less (or in alternativeembodiments, at least 1000, 1500 or 2000 times less) than the IC50 molarconcentration necessary to inhibit to the same degree of CDK2 activityin a standard phosphorylation assay.

As used herein the term “chemotherapy” or “chemotherapeutic agent”refers to treatment with a cytostatic or cytotoxic agent (i.e., acompound) to reduce or eliminate the growth or proliferation ofundesirable cells, for example cancer cells. Thus, as used herein,“chemotherapy” or “chemotherapeutic agent” refers to a cytotoxic orcytostatic agent used to treat a proliferative disorder, for examplecancer.

By “induces G1-arrest” is meant that the inhibitor compound induces aquiescent state in a substantial portion of a cell population at the G1phase of the cell cycle.

By “synchronous reentry into the cell cycle” and similar phrases ismeant that CDK 4/6 replication dependent cells, for example anRb-positive abnormal proliferative cell or hematopoietic stem cell, inG1-arrest due to the effect of a CDK4/6 inhibitor compound reenters thecell-cycle within relatively the same collective timeframe or atrelatively the same rate upon dissipation of the CDK 4/6 inhibitorcompound's effect. Comparatively, by “asynchronous reentry into the cellcycle” is meant that cells in G1 arrest due to the effect of a CDK4/6inhibitor compound reenter the cell cycle within relatively differentcollective timeframes or at relatively different rates upon dissipationof the CDK 4/6 inhibitor compound's effect.

The subject or host treated is typically a human subject, although it isto be understood the methods described herein are effective with respectto other animals, such as mammals and vertebrate species. Moreparticularly, the term subject can include animals used in assays suchas those used in preclinical testing including but not limited to mice,rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine(pigs and hogs), ruminants, equine, poultry, felines, bovines, murines,canines, and the like.

Active Compounds

In one embodiment, the inventions described herein use the CDK4/6inhibitors of Formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof;wherein:Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein z is2, 3 or 4;each X is independently CH or N;each X′ is independently, CH or N;X″ is independently CH₂, S or NH, arranged such that the moiety is astable 5-membered ring;R, R⁸, and R¹¹ are independently H, C₁-C₃ alkyl or haloalkyl, cycloalkylor cycloalkyl containing one or more heteroatoms selected from N, O orS; -(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl,-(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR₃R⁴ any ofwhich may be optionally independently substituted with one or more Rgroups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring;each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl, whereineach of said alkyl, cycloalkyl and haloalkyl groups optionally includesO or N heteroatoms in place of a carbon in the chain and two R¹'s onadjacent ring atoms or on the same ring atom together with the ringatom(s) to which they are attached optionally form a 3-8-membered cycle;y is 0, 1, 2, 3 or 4;R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;-(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0 or 1 and n is 0, 1 or 2;R³ and R⁴ at each occurrence are independently:

-   -   (i) hydrogen or    -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,        cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring; or R³ and R⁴ together with the nitrogen        atom to which they are attached may combine to form a        heterocyclo ring optionally independently substituted with one        or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring;        R⁵ and R⁵* at each occurrence is:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valance;        R^(x) at each occurrence is independently, halo, cyano, nitro,        oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl,        -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,        -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,        -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,        -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,        -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(O)—OR⁵) -(alkylene)_(m)-N(R³)—C(S)—OR⁵,        or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:    -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups        may be further independently substituted with one or more        -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*,        -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*,        -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*,        -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*,        -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*,        -(alkylene)_(m)-C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,        -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,    -   n is 0, 1 or 2, and    -   m is 0 or 1;        R³* and R⁴* at each occurrence are independently:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valance;        or R³* and R⁴* together with the nitrogen atom to which they are        attached may combine to form a heterocyclo ring optionally        independently substituted with one or more R^(x) groups as        allowed by valance; and        R⁶ is H or lower alkyl, -(alkylene)_(m)-heterocyclo,        -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atoms may optionally        combine to form a ring; and        R¹⁰ is (i) NHR^(A), wherein R^(A) is unsubstituted or        substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈        cycloalkyl or cycloalkyl containing one or more heteroatoms        selected from N, O, and S; TT is an unsubstituted or substituted        C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl,        unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted        or substituted C₁-C₆ alkylamino, unsubstituted or substituted        di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl,        unsubstituted or substituted heteroaryl comprising one or two 5-        or 6-member rings and 1-4 heteroatoms selected from N, O and S,        unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted        or substituted heterocycle comprising one or two 5- or 6-member        rings and 1-4 heteroatoms selected from N, O and S; or (ii)        —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³        is R^(A);        or a pharmaceutically acceptable salt, prodrug or isotopic        variant, for example, partically or fully deuterated form        thereof.

In some aspects, the CDK4/6 inhibitor used is of Formula I, Formula II,Formula III, or Formula IV wherein R⁶ is absent.

In some aspects, the CDK4/6 inhibitor used is of Formula III:

and the variables are as defined for compounds of Formulae I and II andpharmaceutically acceptable salts thereof.

In some aspects, R^(x) is not further substituted.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0 or 1 and n is 0, 1 or 2.

In some aspects, R⁸ is hydrogen or C₁-C₃ alkyl.

In some aspects, R is hydrogen or C₁-C₃ alkyl.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ any of which may beoptionally independently substituted with one or more R^(x) groups asallowed by valance, and wherein two R^(x) groups bound to the same oradjacent atom may optionally combine to form a ring.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ without furthersubstitution.

In some aspects, m in R² is 1. In a further aspect, the alkylene in R²is methylene.

In some aspects, R² is

wherein:R^(2*) is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)- or-(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;each R^(x1) is independently-(alkylene)_(m)-(C(O))_(m)-(alkylene)_(m)-(N(R^(N)))_(m)-(alkyl)_(m)wherein each m is independently 0 or 1 provided at least one m is 1,—(C(O))—O-alkyl, -(alkylene)_(m)-cycloalkyl wherein m is 0 or 1,—N(R^(N))-cycloalkyl, —C(O)-cycloalkyl, -(alkylene)_(m)-heterocyclylwherein m is 0 or 1, or —N(R^(N))-heterocyclyl, —C(O)-heterocyclyl,—S(O)₂-(alkylene)_(m) wherein m is 1 or 2, wherein:

-   -   R^(N) is H, C₁ to C₄ alkyl or C₁ to C₆ heteroalkyl, and    -   wherein two R^(x1) can, together with the atoms to which they        attach on P, which may be the same atom, form a ring; and        t is 0, 1 or 2.

In some aspects, each R^(x1) is only optionally substituted byunsubstituted alkyl, halogen or hydroxy.

In some aspects, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl.

In some aspects, at least one R^(x1) is -(alkylene)_(m)-heterocyclylwherein m is 0 or 1.

In some aspects, R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some aspects, R² is

In some aspects, R² is

In some aspects, R² is

wherein:R^(2*) is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)- and-(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;P1 is a 4- to 6-membered monocyclic saturated heterocyclyl group;each R^(x2) is independently hydrogen or alkyl; ands is 0, 1 or 2.

In some aspects, R² is

In some aspects, P1 includes at least one nitrogen.

In some aspects, any alkylene in R²* in any previous aspect is notfurther substituted.

In some aspects, R² is selected from the structures depicted in FIGS.2-4 .

In some aspects, R² is

In some aspects, the compound has general Formula I and morespecifically one of the general structures in FIGS. 5-9 wherein thevariables are as previously defined.

In some aspects, the CDK4/6 inhibitor used has general Formula Ia:

wherein R¹, R², R and y are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ia and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Ia and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl andR^(2*) is as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ib:

wherein R² and R are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ib and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Ib and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ic:

wherein R² and R are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ic and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Ic and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Id:

wherein R² and R are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Id and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Id and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ie:

In some embodiments, the CDK4/6 inhibitor used has Formula Ie and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Ie and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Je and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula If:

In some embodiments, the CDK4/6 inhibitor used has Formula If and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula If and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ig:

In some embodiments, the CDK4/6 inhibitor used has Formula Ig and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Ig and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ih:

In some embodiments, the CDK4/6 inhibitor used has Formula Ih and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Ih and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ii:

In some embodiments, the CDK4/6 inhibitor used has Formula Ii and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Ii and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the CDK4/6 inhibitor used has Formula Ij:

In some embodiments, the CDK4/6 inhibitor used has Formula Ij and R isalkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Ij and R isH.

In some embodiments, the CDK4/6 inhibitor used has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Ij and R isH, and both X are N.

In some embodiments, the CDK4/6 inhibitor used has the structure:

In some embodiments, the CDK4/6 inhibitor used has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Il:

In some embodiments, the CDK4/6 inhibitor used has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula Im:

In some embodiments, the CDK4/6 inhibitor used has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula IIa:

In some embodiments, the CDK4/6 inhibitor used has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the CDK4/6 inhibitor used has Formula IIb:

In some embodiments, the CDK4/6 inhibitor used has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the CDK4/6 inhibitor used has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some aspects, the CDK4/6 inhibitor used is:

Further specific CDK4/6 inhibitor compounds that fall within the presentinvention and that can be used in the disclosed methods of treatment andcombinations include the structures listed in Table 1 below.

TABLE 1 Nonlimiting Examples of Compounds of Formula I, II, III, IV, orV Structure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

QQQ

RRR

SSS

TTT

UUU

VVV

WWW

XXX

The CDK4/6 inhibitors for use in the described methods are highlyselective, potent CDK4/6 inhibitors, with minimal CDK2 inhibitoryactivity. In a range of embodiments, a compound for use in the methodsdescribed herein has a CDK4/CycD1 IC50 inhibitory concentration valuethat is >1500 times, >1800 times, >2000 times, >2200 times, >2500times, >2700 times, >3000 times, >3200 times or greater lower than itsrespective IC50 concentration value for CDK2/CycE inhibition. In anotherrange of embodiments, a compound for use in the methods described hereinhas an IC50 concentration value for CDK4/CycD1 inhibition that is about<1.50 nM, <1.25 nM, <1.0 nM, <0.90 nM, <0.85 nM, <0.80 nM, <0.75 nM,<0.70 nM, <0.65 nM, <0.60 nM, <0.55 nM, or less. In yet a range ofembodiments, a CDK4/6 inhibitor for use in the methods described hereinhas an IC50 concentration value for CDK2/CycE inhibition that isabout >1.0 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50μM, >2.75 μM, >3.0 μM, >3.25 μM, >3.5 μM or greater. In still otherembodiments, a compound for use in the methods described herein has anIC50 concentration value for CDK2/CycA IC50 that is >0.80 μM, >0.85μM, >0.90 μM, >0.95 μM, >0.1.0 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0μM, >2.25 μM, >2.50 μM, >2.75 uM, >3.0 μM or greater.

Isotopic Substitution

In certain aspects of the present invention, included is the use ofCDK4/6 inhibitors and/or a chemotherapeutic agent with desired isotopicsubstitutions of atoms, at amounts above the natural abundance of theisotope, i.e., enriched. Isotopes are atoms having the same atomicnumber but different mass numbers, i.e., the same number of protons buta different number of neutrons. By way of general example and withoutlimitation, isotopes of hydrogen, for example, deuterium (²H) andtritium (³H) may be used anywhere in described structures. Alternativelyor in addition, isotopes of carbon, e.g., ¹³C and ¹⁴C, may be used. Apreferred isotopic substitution is deuterium for hydrogen at one or morelocations on the molecule to improve the performance of the drug. Thedeuterium can be bound in a location of bond breakage during metabolism(an α-deuterium kinetic isotope effect) or next to or near the site ofbond breakage (a β-deuterium kinetic isotope effect).

Substitution with isotopes such as deuterium can afford certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements. Substitution of deuterium for hydrogen at a site ofmetabolic break down can reduce the rate of or eliminate the metabolismat that bond. At any position of the compound that a hydrogen atom maybe present, the hydrogen atom can be any isotope of hydrogen, includingprotium (¹H), deuterium (²H) and tritium (³H). Thus, reference herein toa compound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

The term “isotopically-labeled” analog refers to an analog that is a“deuterated analog”, a “¹³C-labeled analog,” or a“deuterated/¹³C-labeled analog.” The term “deuterated analog” means acompound described herein, whereby a H-isotope, i.e., hydrogen/protium(H), is substituted by a H-isotope, i.e., deuterium (²H). Deuteriumsubstitution can be partial or complete. Partial deuterium substitutionmeans that at least one hydrogen is substituted by at least onedeuterium. In certain embodiments, the isotope is 90, 95 or 99% or moreenriched in an isotope at any location of interest. In some embodimentsit is deuterium that is 90, 95 or 99% enriched at a desired location.

Further specific compounds that fall within the present invention andthat can be used in the disclosed methods of treatment and compositionsinclude the structures of Formula I, II, III, IV, or V, and those listedabove in Table 1.

Combination Therapy

In one aspect, provided is a treatment regimen comprising theadministration of a CDK4/6 inhibitor described herein in combination orin alternation with at least one additional chemotherapeutic agent. Thecombinations and/or alternations disclosed herein can be administeredfor beneficial, additive, or synergistic effect in the treatment ofRb-positive abnormal cellular proliferative disorders.

In specific embodiments, the treatment regimen includes theadministration of a CDK4/6 inhibitor described herein in combination oralternation with at least one additional kinase inhibitor. In oneembodiment, the at least one additional kinase inhibitor is selectedfrom a phosphoinositide 3-kinase (PI3K) inhibitor, a Bruton's tyrosinekinase (BTK) inhibitor, or a spleen tyrosine kinase (Syk) inhibitor, ora combination thereof.

PI3K inhibitors that may be used in the present invention are wellknown. Examples of PI3 kinase inhibitors include but are not limited toWortmannin, demethoxyviridin, perifosine, idelalisib, Pictilisib,Palomid 529, ZSTK474, PWT33597, CUDC-907, and AEZS-136, duvelisib,GS-9820, BKM120, GDC-0032 (Taselisib)(2-[4-[2-(2-Isopropyl-5-methyl-1,2,4-triazol-3-yl)-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepin-9-yl]pyrazol-1-yl]-2-methylpropanamide),MLN-1117 ((2R)-1-Phenoxy-2-butanyl hydrogen (S)-methylphosphonate; orMethyl(oxo) {[(2R)-1-phenoxy-2-butanyl]oxy}phosphonium)), BYL-719((2S)—N1-[4-Methyl-5-[2-(2,2,2-trifluoro-1,1-dimethylethyl)-4-pyridinyl]-2-thiazolyl]-1,2-pyrrolidinedicarboxamide),GSK2126458(2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide)(omipalisib), TGX-221((±)-7-Methyl-2-(morpholin-4-yl)-9-(1-phenylaminoethyl)-pyrido[1,2-a]-pyrimidin-4-one),GSK2636771(2-Methyl-1-(2-methyl-3-(trifluoromethyl)benzyl)-6-morpholino-lH-benzo[d]imidazole-4-carboxylicacid dihydrochloride), KIN-193((R)-2-((1-(7-methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)ethyl)amino)benzoicacid), TGR-1202/RP5264, GS-9820((S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-mohydroxypropan-1-one),GS-1101(5-fluoro-3-phenyl-2-([S)]-1-[9H-purin-6-ylamino]-propyl)-3H-quinazolin-4-one),AMG-319, GSK-2269557, SAR245409(N-(4-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxalin-2-yl)sulfamoyl)phenyl)-3-methoxy-4methylbenzamide), BAY80-6946(2-amino-N-(7-methoxy-8-(3-morpholinopropoxy)-2,3-dihydroimidazo[1,2-c]quinaz),AS 252424(5-[1-[5-(4-Fluoro-2-hydroxy-phenyl)-furan-2-yl]-meth-(Z)-ylidene]-thiazolidine-2,4-dione),CZ 24832(5-(2-amino-8-fluoro-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-tert-butylpyridine-3-sulfonamide),Buparlisib(5-[2,6-Di(4-morpholinyl)-4-pyrimidinyl]-4-(trifluoromethyl)-2-pyridinamine),GDC-0941(2-(lH-Indazol-4-yl)-6-[[4-(methylsulfonyl)-1-piperazinyl]methyl]-4-(4-morpholinyl)thieno[3,2-d]pyrimidine),GDC-0980((S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6yl)methyl)piperazin-l-yl)-2-hydroxypropan-l-one (also known as RG7422)),SF1126((8S,14S,17S)-14-(carboxymethyl)-8-(3-guanidinopropyl)-17-(hydroxymethyl)-3,6,9,12,15-pentaoxo-1-(4-(4-oxo-8-phenyl-4H-chromen-2-yl)morpholino-4-ium)-2-oxa-7,10,13,16-tetraazaoctadecan-18-oate),PF-05212384(N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N′-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea)(gedatolisib), LY3023414, BEZ235(2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-lH-imidazo[4,5-c]quinolin-l-yl]phenyl}propanenitrile)(dactolisib), XL-765(N-(3-(N-(3-(3,5-dimethoxyphenylamino)quinoxalin-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide),and GSK1059615(5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidenedione),PX886([(3aR,6E,9S,9aR,10R,11aS)-6-[[bis(prop-2-enyl)amino]methylidene]-5-hydroxy-9-(methoxymethyl)-9a,11a-dimethyl-1,4,7-trioxo-2,3,3a,9,10,11-hexahydroindeno[4,5h]isochromen-10-yl]acetate (also known as sonolisib)), LY294002, AZD8186, PF-4989216,pilaralisib, GNE-317, PI-3065, PI-103, NU7441 (KU-57788), HS 173,VS-5584 (SB2343), CZC24832, TG100-115, A66, YM201636, CAY10505, PIK-75,PIK-93, AS-605240, BGT226 (NVP-BGT226), AZD6482, voxtalisib, alpelisib,IC-87114, TGI100713, CH5132799, PKI-402, copanlisib (BAY 80-6946), XL147, PIK-90, PIK-293, PIK-294, 3-MA (3-methyladenine), AS-252424,AS-604850, apitolisib (GDC-0980; RG7422), and the structure described inWO2014/071109 having the formula:

In a particular embodiment, the CDK4/6 inhibitors described herein areadministered in combination or alternation with pictilisib, and the hostis suffering from a cancer selected from colorectal, breast, soft tissuesarcoma, melanoma, ovarian, gastric, or prostate. In one embodiment, thecancer is breast cancer.

In one embodiment, the CDK4/6 inhibitor is combined in a single dosageform with the PIk3 inhibitor.

BTK inhibitors for use in the present invention are well known. Examplesof BTK inhibitors include ibrutinib (also known asPCI-32765)(Imbruvica™)(1-[(3R)-3-[4-amino-3-(4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one),dianilinopyrimidine-based inhibitors such as AVL-101 and AVL-291/292(N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide)(Avila Therapeutics) (see US Patent Publication No 2011/0117073,incorporated herein in its entirety), Dasatinib([N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide],LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-ibromophenyl)propenamide), GDC-0834([R—N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide],CGI-5604-(tert-butyl)-N-(3-(8-(phenylamino)imidazo[1,2-a]pyrazin-6-yl)phenyl)benzamide,CGI-1746(4-(tert-butyl)-N-(2-methyl-3-(4-methyl-6-((4-(morpholine-4-carbonyl)phenyl)amino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)benzamide),CNX-774(4-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-yl)amino)phenoxy)-N-methylpicolinamide),CTA056(7-benzyl-1-(3-(piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one),GDC-0834((R)—N-(3-(6-((4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenyl)amino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide),GDC-0837((R)—N-(3-(6-((4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenyl)amino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide),HM-71224, ACP-196, ONO-4059 (Ono Pharmaceuticals), PRT062607(4-((3-(2H-1,2,3-triazol-2-yl)phenyl)amino)-2-(((1R,2S)-2-aminocyclohexyl)amino)pyrimidine-5-carboxamidehydrochloride), QL-47(1-(1-acryloylindolin-6-yl)-9-(1-methyl-1H-pyrazol-4-yl)benzo[h][1,6]naphthyridin-2(1H)-one),and RN486(6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one),and other molecules capable of inhibiting BTK activity, for examplethose BTK inhibitors disclosed in Akinleye et ah, Journal of Hematology& Oncology, 2013, 6:59, the entirety of which is incorporated herein byreference. In one embodiment, the CDK4/6 inhibitor is combined in asingle dosage form with the BTK inhibitor.

Syk inhibitors for use in the present invention are well known, andinclude, for example, Cerdulatinib(4-(cyclopropylamino)-2-((4-(4-(ethylsulfonyl)piperazin-1-yl)phenyl)amino)pyrimidine-5-carboxamide),entospletinib(6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine),fostamatinib([6-({5-Fluoro-2-[(3,4,5-trimethoxyphenyl)amino]-4-pyrimidinyl}amino)-2,2-dimethyl-3-oxo-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazin-4-yl]methyldihydrogen phosphate), fostamatinib disodium salt (sodium(6-((5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2-dimethyl-3-oxo-2H-pyrido[3,2-b][1,4]oxazin-4(3H)-yl)methylphosphate), BAY 61-3606(2-(7-(3,4-Dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino)-nicotinamideHCl), R09021(6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylicacid amide), imatinib (Gleevac;4-[(4-methylpiperazin-1-yl)methyl]-N-(4-methyl-3-{[4-(pyridin-3-yl)pyrimidin-2-yl]amino}phenyl)benzamide),staurosporine, GSK143(2-(((3R,4R)-3-aminotetrahydro-2H-pyran-4-yl)amino)-4-(p-tolylamino)pyrimidine-5-carboxamide),PP2(1-(tert-butyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine),PRT-060318(2-(((1R,2S)-2-aminocyclohexyl)amino)-4-(m-tolylamino)pyrimidine-5-carboxamide),PRT-062607(4-((3-(2H-1,2,3-triazol-2-yl)phenyl)amino)-2-(((1R,2S)-2-aminocyclohexyl)amino)pyrimidine-5-carboxamidehydrochloride), R112(3,3′-((5-fluoropyrimidine-2,4-diyl)bis(azanediyl))diphenol), R348(3-Ethyl-4-methylpyridine), R406(6-((5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one),piceatannol (3-Hydroxyresveratol), YM193306 (see Singh et al. Discoveryand Development of Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med.Chem. 2012, 55, 3614-3643), 7-azaindole, piceatannol, ER-27319 (seeSingh et al. Discovery and Development of Spleen Tyrosine Kinase (SYK)Inhibitors, J. Med. Chem. 2012, 55, 3614-3643 incorporated in itsentirety herein), Compound D (see Singh et al. Discovery and Developmentof Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med. Chem. 2012, 55,3614-3643 incorporated in its entirety herein), PRT060318 (see Singh etal. Discovery and Development of Spleen Tyrosine Kinase (SYK)Inhibitors, J. Med. Chem. 2012, 55, 3614-3643 incorporated in itsentirety herein), luteolin (see Singh et al. Discovery and Developmentof Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med. Chem. 2012, 55,3614-3643 incorporated in its entirety herein), apigenin (see Singh etal. Discovery and Development of Spleen Tyrosine Kinase (SYK)Inhibitors, J. Med. Chem. 2012, 55, 3614-3643 incorporated in itsentirety herein), quercetin (see Singh et al. Discovery and Developmentof Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med. Chem. 2012, 55,3614-3643 incorporated in its entirety herein), fisetin (see Singh etal. Discovery and Development of Spleen Tyrosine Kinase (SYK)Inhibitors, J. Med. Chem. 2012, 55, 3614-3643 incorporated in itsentirety herein), myricetin (see Singh et al. Discovery and Developmentof Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med. Chem. 2012, 55,3614-3643 incorporated in its entirety herein), morin (see Singh et al.Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors, J.Med. Chem. 2012, 55, 3614-3643 incorporated in its entirety herein). Inone embodiment, the CDK4/6 inhibitor is combined in a single dosage formwith the Syk inhibitor.

MEK inhibitors for use in the present invention are well known, andinclude, for example, trametinib/GSK1120212(N-(3-{3-Cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-l(2H-yl}phenyl)acetamide),selumetinib(6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide),pimasertib/AS703026/MSC 1935369((S)—N-(2,3-dihydroxypropyl)-3-((2-fluoro-4-iodophenyl)amino)isonicotinamide),XL-518/GDC-0973(1-({3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl}carbonyl)-3-[(2S)-piperidin-2-yl]azetidin-3-ol),refametinib/BAY869766/RDEAl 19(N-(3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-6-methoxyphenyl)-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide),PD-0325901(N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide),TAK733((R)-3-(2,3-Dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione),MEK162/ARRY438162(5-[(4-Bromo-2-fluorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6-carboxamide),R05126766(3-[[3-Fluoro-2-(methylsulfamoylamino)-4-pyridyl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one),WX-554, R04987655/CH4987655(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)-5-((3-oxo-1,2-oxazinan-2yl)methyl)benzamide),or AZD8330 (2-((2-fluoro-4-iodophenyl)amino)-N-(2hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide),U0126-EtOH, PD184352 (CI-1040), GDC-0623, BI-847325, cobimetinib,PD98059, BIX 02189, BIX 02188, binimetinib, SL-327, TAK-733, PD318088,and additional MEK inhibitors as described below. In one embodiment, theCDK4/6 inhibitor is combined in a single dosage form with the MEKinhibitor.

Raf inhibitors for use in the present invention are well known, andinclude, for example, Vemurafinib(N-[3-[[5-(4-Chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl]-2,4-difluorophenyl]-1-propanesulfonamide),sorafenib tosylate(4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methylpyridine-2-carboxamide;4-methylbenzenesulfonate), AZ628(3-(2-cyanopropan-2-yl)-N-(4-methyl-3-(3-methyl-4-oxo-3,4-dihydroquinazolin-6-ylamino)phenyl)benzamide),NVP-BHG712(4-methyl-3-(1-methyl-6-(pyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)-N-(3-(trifluoromethyl)phenyl)benzamide),RAF-265(1-methyl-5-[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]pyridin-4-yl]oxy-N-[4-(trifluoromethyl)phenyl]benzimidazol-2-amine),2-Bromoaldisine(2-Bromo-6,7-dihydro-1H,5H-pyrrolo[2,3-c]azepine-4,8-dione), Raf KinaseInhibitor IV(2-chloro-5-(2-phenyl-5-(pyridin-4-yl)-1H-imidazol-4-yl)phenol),Sorafenib N-Oxide(4-[4-[[[[4-Chloro-3(trifluoroMethyl)phenyl]aMino]carbonyl]aMino]phenoxy]-N-Methyl-2pyridinecarboxaMide1-Oxide), PLX-4720, dabrafenib (GSK2118436), GDC-0879, RAF265, AZ 628,Sf590885, ZM336372, GW5074, TAK-632, CEP-32496, LY3009120, and GX818(Encorafenib). In one embodiment, the CDK4/6 inhibitor is combined in asingle dosage form with the Raf inhibitor.

In one embodiment, the at least one additional chemotherapeutic agentcombined or alternated with the CDK4/6 inhibitor is a programmed deathprotein 1 (PD-1) inhibitor or programmed death protein ligand 1 or 2inhibitor. PD-1 inhibitors are known in the art, and include, forexample, nivolumab (BMS), pembrolizumab (Merck), pidilizumab(CureTech/Teva), AMP-244 (Amplimmune/GSK), BMS-936559 (BMS), andMEDI4736 (Roche/Genentech), and MPDL3280A (Genentech). In oneembodiment, the CDK4/6 inhibitor is combined in a single dosage formwith the PD-1 inhibitor.

In one embodiment, the at least one additional chemotherapeutic agentcombined or alternated with the CDK4/6 inhibitor is a B-cell lymphoma 2(Bcl-2) protein inhibitor. BCL-2 inhibitors are known in the art, andinclude, for example, ABT-199(4-[4-[[2-(4-Chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl]piperazin-1-yl]-N-[[3-nitro-4-[[(tetrahydro-2H-pyran-4-yl)methyl]amino]phenyl]sulfonyl]-2-[(1H-pyrrolo[2,3-b]pyridin-5-yl)oxy]benzamide),ABT-737(4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide) (navitoclax), ABT-263((R)-4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholino-1-(phenylthio)butan-2-yl)amino)-3((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide),GX15-070 (obatoclax mesylate,(2Z)-2-[(5Z)-5-[(3,5-dimethyl-lH-pyrrol-2-yl)methylidene]-4-methoxypyrrol-2-ylidene]indole;methanesulfonic acid))), 2-methoxy-antimycin A3, YC137(4-(4,9-dioxo-4,9-dihydronaphtho[2,3-d]thiazol-2-ylamino)-phenyl ester),pogosin, ethyl2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate,Nilotinib-d3, TW-37(N-[4-[[2-(1,1-Dimethylethyl)phenyl]sulfonyl]phenyl]-2,3,4-trihydroxy-5-[[2-(1-methylethyl)phenyl]methyl]benzamide),Apogossypolone (ApoG2), HA14-1, AT101, sabutoclax, gambogic acid, orG3139 (Oblimersen). In one embodiment, the CDK4/6 inhibitor is combinedin a single dosage form with the at least one BCL-2 inhibitor.

Additional Combinations/Alternations

In one embodiment, a CDK4/6 inhibitor combination or alternation asdescribed above, that is a CDK4/6 inhibitor as described herein combinedor alternated with a kinase inhibitor, PD-1 inhibitor, or BCL-2inhibitor, can be further combined with an additional therapeutic totreat the Rb-positive cancer. The second therapy can be animmunotherapy. As discussed in more detail below, the CDK4/6 inhibitoror combination agent can be conjugated to an antibody, radioactiveagent, or other targeting agent that directs the CDK4/6 inhibitor to thediseased or abnormally proliferating cell. In another embodiment, theCDK4/6 inhibitor combination is used in combination with anotherpharmaceutical or a biologic agent (for example an antibody) to increasethe efficacy of treatment with a combined or a synergistic approach. Inan embodiment, CDK4/6 inhibitor combination can be used with T-cellvaccination, which typically involves immunization with inactivatedautoreactive T cells to eliminate an Rb-positive cancer cell populationas described herein. In another embodiment, the CDK4/6 inhibitorcombination is used in combination with a bispecific T-cell Engager(BiTE), which is an antibody designed to simultaneously bind to specificantigens on endogenous T cells and Rb-positive cancer cells as describedherein, linking the two types of cells.

In one embodiment, the additional therapy is a monoclonal antibody(MAb). Some MAbs stimulate an immune response that destroys cancercells. Similar to the antibodies produced naturally by B cells, theseMAbs “coat” the cancer cell surface, triggering its destruction by theimmune system. For example, bevacizumab targets vascular endothelialgrowth factor (VEGF), a protein secreted by tumor cells and other cellsin the tumor's microenvironment that promotes the development of tumorblood vessels. When bound to bevacizumab, VEGF cannot interact with itscellular receptor, preventing the signaling that leads to the growth ofnew blood vessels. Similarly, cetuximab and panitumumab target theepidermal growth factor receptor (EGFR), and trastuzumab targets thehuman epidermal growth factor receptor 2 (HER-2). MAbs that bind to cellsurface growth factor receptors prevent the targeted receptors fromsending their normal growth-promoting signals. They may also triggerapoptosis and activate the immune system to destroy tumor cells.

Another group of cancer therapeutic MAbs are the immunoconjugates. TheseMAbs, which are sometimes called immunotoxins or antibody-drugconjugates, consist of an antibody attached to a cell-killing substance,such as a plant or bacterial toxin, a chemotherapy drug, or aradioactive molecule. The antibody latches onto its specific antigen onthe surface of a cancer cell, and the cell-killing substance is taken upby the cell. FDA-approved conjugated MAbs that work this way includeado-trastuzumab emtansine, which targets the HER-2 molecule to deliverthe drug DM1, which inhibits cell proliferation, to HER-2 expressingmetastatic breast cancer cells.

Immunotherapies with T cells engineered to recognize cancer cells viabispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) areapproaches with potential to ablate both dividing and non/slow-dividingsubpopulations of cancer cells.

Bispecific antibodies, by simultaneously recognizing target antigen andan activating receptor on the surface of an immune effector cell, offeran opportunity to redirect immune effector cells to kill cancer cells.The other approach is the generation of chimeric antigen receptors byfusing extracellular antibodies to intracellular signaling domains.Chimeric antigen receptor-engineered T cells are able to specificallykill tumor cells in a MHC-independent way.

In some embodiments, the CDK4/6 inhibitor combination or alternationdescribed herein can be administered to the subject in furthercombination or alternation with other chemotherapeutic agents. Ifconvenient, the CDK4/6 inhibitor combination or alternation describedherein can be administered at the same time as another chemotherapeuticagent, in order to simplify the treatment regimen. In some embodiments,the CDK4/6 inhibitor combination and the other chemotherapeutic can beprovided in a single formulation. In one embodiment, the use of theCDK4/6 inhibitor combination described herein is combined in atherapeutic regime with other agents. Such agents may include, but arenot limited to, at least one of tamoxifen, midazolam, letrozole,bortezomib, anastrozole, goserelin, an mTOR inhibitor, a PI3 kinaseinhibitor as described above, a dual mTOR-PI3K inhibitor, a MEKinhibitor, a RAS inhibitor, ALK inhibitor, an HSP inhibitor (forexample, HSP70 and HSP 90 inhibitor, or a combination thereof), a BCL-2inhibitor as described above, apopototic inducing compounds, an AKTinhibitor, including but not limited to, MK-2206, GSK690693, Perifosine,(KRX-0401), GDC-0068, Triciribine, AZD5363, Honokiol, PF-04691502, andMiltefosine, a PD-1 inhibitor as described above including but notlimited to, Nivolumab, CT-011, MK-3475, BMS936558, and AMP-514 or aFLT-3 inhibitor, including but not limited to, P406, Dovitinib,Quizartinib (AC220), Amuvatinib (MP-470), Tandutinib (MLN518),ENMD-2076, and KW-2449, or a combination thereof. Examples of mTORinhibitors include but are not limited to rapamycin and its analogs,everolimus (Afinitor), temsirolimus, ridaforolimus, sirolimus, anddeforolimus. Examples of MEK inhibitors include but are not limited totametinib/GSK1120212(N-(3-{3-Cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H-yl}phenyl)acetamide),selumetinob(6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide),pimasertib/AS703026/MSC1935369((S)—N-(2,3-dihydroxypropyl)-3-((2-fluoro-4-iodophenyl)amino)isonicotinamide),XL-518/GDC-0973(1-({3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl}carbonyl)-3-[(2S)-piperidin-2-yl]azetidin-3-ol)(cobimetinib), refametinib/BAY869766/RDEA119(N-(3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-6-methoxyphenyl)-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide),PD-0325901(N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide),TAK733((R)-3-(2,3-Dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3d]pyrimidine-4,7(3H,8H)-dione),MEK162/ARRY438162(5-[(4-Bromo-2-fluorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6carboxamide), R05126766(3-[[3-Fluoro-2-(methylsulfamoylamino)-4-pyridyl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one),WX-554, R04987655/CH4987655(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)-5-((3-oxo-1,2-oxazinan-2yl)methyl)benzamide), or AZD8330(2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide).Examples of RAS inhibitors include but are not limited to Reolysin andsiG12D LODER. Examples of ALK inhibitors include but are not limited toCrizotinib, Ceritinib (Zykadia), AP26113, and LDK378. HSP inhibitorsinclude but are not limited to Geldanamycin or17-N-Allylamino-17-demethoxygeldanamycin (17AAG), and Radicicol. In aparticular embodiment, a compound described herein is administered incombination with letrozole and/or tamoxifen. Other chemotherapeuticagents that can be used in combination with the compounds describedherein include, but are not limited to, chemotherapeutic agents that donot require cell cycle activity for their anti-neoplastic effect.

In one embodiment, a CDK4/6 inhibitor combination described herein canbe combined with a chemotherapeutic selected from, but are not limitedto, Imatinib mesylate (Gleevac®), Dasatinib (Sprycel®), Nilotinib(Tasigna®), Bosutinib (Bosulif®), Trastuzumab (Herceptin®),trastuzumab-DM1, Pertuzumab (Perjeta™), Lapatinib (Tykerb®), Gefitinib(Iressa®), Erlotinib (Tarceva®), Cetuximab (Erbitux®), Panitumumab(Vectibix®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®), Vorinostat(Zolinza®), Romidepsin (Istodax®), Bexarotene (Tagretin®), Alitretinoin(Panretin®), Tretinoin (Vesanoid®), Carfilizomib (Kyprolis™),Pralatrexate (Folotyn®), Bevacizumab (Avastin®), Ziv-aflibercept(Zaltrap®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Pazopanib(Votrient®), Regorafenib (Stivarga®), and Cabozantinib (Cometriq™).

In certain aspects, the additional therapeutic agent is ananti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic,an additional therapeutic agent, or an immunosuppressive agent.

Suitable chemotherapeutic agents include, but are not limited to, aradioactive molecule, a toxin, also referred to as cytotoxin orcytotoxic agent, which includes any agent that is detrimental to theviability of cells, and liposomes or other vesicles containingchemotherapeutic compounds. General anticancer pharmaceutical agentsinclude: Vincristine (Oncovin®) or liposomal vincristine (Marqibo®),Daunorubicin (daunomycin or Cerubidine®) or doxorubicin (Adriamycin®),Cytarabine (cytosine arabinoside, ara-C, or Cytosar®), L-asparaginase(Elspar®) or PEG-L-asparaginase (pegaspargase or Oncaspar®), Etoposide(VP-16), Teniposide (Vumon®), 6-mercaptopurine (6-MP or Purinethol®),Methotrexate, Cyclophosphamide (Cytoxan®), Prednisone, Dexamethasone(Decadron), imatinib (Gleevec®), dasatinib (Sprycel®), nilotinib(Tasigna®), bosutinib (Bosulif®), and ponatinib (Iclusig™). Examples ofadditional suitable chemotherapeutic agents include but are not limitedto 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine,6-thioguanine, actinomycin D, adriamycin, aldesleukin, an alkylatingagent, allopurinol sodium, altretamine, amifostine, anastrozole,anthramycin (AMC)), an anti-mitotic agent, cis-dichlorodiamine platinum(II) (DDP) cisplatin), diamino dichloro platinum, anthracycline, anantibiotic, an antimetabolite, asparaginase, BCG live (intravesical),betamethasone sodium phosphate and betamethasone acetate, bicalutamide,bleomycin sulfate, busulfan, calcium leucouorin, calicheamicin,capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU),Chlorambucil, Cisplatin, Cladribine, Colchicin, conjugated estrogens,Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine, cytochalasinB, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin (formerlyactinomycin), daunirubicin HCL, daunorucbicin citrate, denileukindiftitox, Dexrazoxane, Dibromomannitol, dihydroxy anthracin dione,Docetaxel, dolasetron mesylate, doxorubicin HCL, dronabinol, E. coliL-asparaginase, emetine, epoetin-α, Erwinia L-asparaginase, esterifiedestrogens, estradiol, estramustine phosphate sodium, ethidium bromide,ethinyl estradiol, etidronate, etoposide citrororum factor, etoposidephosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate,fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoids,goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea,idarubicin HCL, ifosfamide, interferon α-2b, irinotecan HCL, letrozole,leucovorin calcium, leuprolide acetate, levamisole HCL, lidocaine,lomustine, maytansinoid, mechlorethamine HCL, medroxyprogesteroneacetate, megestrol acetate, melphalan HCL, mercaptipurine, mesna,methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane,mitoxantrone, nilutamide, octreotide acetate, ondansetron HCL,paclitaxel, pamidronate disodium, pentostatin, pilocarpine HCL,plimycin, polifeprosan 20 with carmustine implant, porfimer sodium,procaine, procarbazine HCL, propranolol, rituximab, sargramostim,streptozotocin, tamoxifen, taxol, teniposide, tenoposide, testolactone,tetracaine, thioepa chlorambucil, thioguanine, thiotepa, topotecan HCL,toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastinesulfate, vincristine sulfate, and vinorelbine tartrate.

Additional therapeutic agents that can be administered in combinationwith a CDK4/6 inhibitor combination disclosed herein can includebevacizumab, sutinib, sorafenib, 2-methoxyestradiol or 2ME2, finasunate,vatalanib, vandetanib, aflibercept, volociximab, etaracizumab(MEDI-522), cilengitide, erlotinib, cetuximab, panitumumab, gefitinib,trastuzumab, dovitinib, figitumumab, atacicept, rituximab, alemtuzumab,aldesleukine, atlizumab, tocilizumab, temsirolimus, everolimus,lucatumumab, dacetuzumab, HLL1, huN901-DM1, atiprimod, natalizumab,bortezomib, carfilzomib, marizomib, tanespimycin, saquinavir mesylate,ritonavir, nelfinavir mesylate, indinavir sulfate, belinostat,panobinostat, mapatumumab, lexatumumab, dulanermin, ABT-737, oblimersen,plitidepsin, talmapimod, P276-00, enzastaurin, tipifarnib, perifosine,imatinib, dasatinib, lenalidomide, thalidomide, simvastatin, celecoxib,bazedoxifene, AZD4547, rilotumumab, oxaliplatin (Eloxatin), PD0332991,ribociclib (LEE011), amebaciclib (LY2835219), HDM201, fulvestrant(Faslodex), exemestane (Aromasin), PIM447, ruxolitinib (INC424), BGJ398,necitumumab, pemetrexed (Alimta), and ramucirumab (IMC-1121B).

In one aspect of the present invention, a CDK4/6 inhibitor combinationdescribed herein can be combined with at least one immunosuppressiveagent. The immunosuppressive agent is preferably selected from the groupconsisting of a calcineurin inhibitor, e.g. a cyclosporin or anascomycin, e.g. Cyclosporin A (NEORAL®), FK506 (tacrolimus),pimecrolimus, a mTOR inhibitor, e.g. rapamycin or a derivative thereof,e.g. Sirolimus (RAPAMUNE®), Everolimus (Certican®), temsirolimus,zotarolimus, biolimus-7, biolimus-9, a rapalog, e.g.ridaforolimus,azathioprine, campath 1H, a SiP receptor modulator, e.g. fingolimod oran analogue thereof, an anti IL-8 antibody, mycophenolic acid or a saltthereof, e.g. sodium salt, or a prodrug thereof, e.g. MycophenolateMofetil (CELLCEPT®), OKT3 (ORTHOCLONE OKT3@), Prednisone, ATGAM®,THYMOGLOBULIN®, Brequinar Sodium, OKT4, T10B9.A-3A, 33B3.1,15-deoxyspergualin, tresperimus, Leflunomide ARAVA®, CTLAI-Ig,anti-CD25, anti-IL2R, Basiliximab (SVIMULECT®), Daclizumab (ZENAPAX®),mizorbine, methotrexate, dexamethasone, ISAtx-247, SDZ ASM 981(pimecrolimus, Elidel®), CTLA4lg (Abatacept), belatacept, LFA3lg,etanercept (sold as Enbrel® by Immunex), adalimumab (Humira®),infliximab (Remicade®), an anti-LFA-1 antibody, natalizumab (Antegren®),Enlimomab, gavilimomab, antithymocyte immunoglobulin, siplizumab,Alefacept efalizumab, pentasa, mesalazine, asacol, codeine phosphate,benorylate, fenbufen, naprosyn, diclofenac, etodolac and indomethacin,aspirin and ibuprofen.

In certain embodiments, a CDK4/6 inhibitor combination described hereinis administered to the subject prior to treatment with anotherchemotherapeutic agent, during treatment with another chemotherapeuticagent, after administration of another chemotherapeutic agent, or acombination thereof.

In some embodiments, the selective CDK4/6 inhibitor combination can beadministered to the subject such that the other chemotherapeutic agentcan be administered either at higher doses (increased chemotherapeuticdose intensity) or more frequently (increased chemotherapeutic dosedensity). Dose-dense chemotherapy is a chemotherapy treatment plan inwhich drugs are given with less time between treatments than in astandard chemotherapy treatment plan. Chemotherapy dose intensityrepresents unit dose of chemotherapy administered per unit time. Doseintensity can be increased or decreased through altering doseadministered, time interval of administration, or both.

In one embodiment of the invention, the CDK4/6 inhibitor combinationdescribed herein can be administered in a concerted regimen with anotheragent such as a non-DNA-damaging, targeted anti-neoplastic agent or ahematopoietic growth factor agent. It has recently been reported thatthe untimely administration of hematopoietic growth factors can haveserious side effects. For example, the use of the EPO family of growthfactors has been associated with arterial hypertension, cerebralconvulsions, hypertensive encephalopathy, thromboembolism, irondeficiency, influenza like syndromes and venous thrombosis. The G-CSFfamily of growth factors has been associated with spleen enlargement andrupture, respiratory distress syndrome, allergic reactions and sicklecell complications. By combining the administration of the CDK4/6inhibitor combination as described herein with the timely administrationof hematopoietic growth factors, for example, at the time point whereinthe affected cells are no longer under growth arrest, it is possible forthe health care practitioner to decrease the amount of the growth factorto minimize the unwanted adverse effects while achieving the desiredtherapeutic benefit. As such, in one embodiment, the use of the CDK4/6inhibitor combination or methods described herein is combined with theuse of hematopoietic growth factors including, but not limited to,granulocyte colony stimulating factor (G-CSF, for example, sold asNeupogen (filgrastin), Neulasta (peg-filgrastin), or lenograstin),granulocyte-macrophage colony stimulating factor (GM-CSF, for examplesold as molgramostim and sargramostim (Leukine)), M-CSF (macrophagecolony stimulating factor), thrombopoietin (megakaryocyte growthdevelopment factor (MGDF), for example sold as Romiplostim andEltrombopag) interleukin (IL)-12, interleukin-3, interleukin-11(adipogenesis inhibiting factor or oprelvekin), SCF (stem cell factor,steel factor, kit-ligand, or KL) and erythropoietin (EPO), and theirderivatives (sold as for example epoetin-α as Darbopoetin, Epocept,Nanokine, Epofit, Epogin, Eprex and Procrit; epoetin-β sold as forexample NeoRecormon, Recormon and Micera), epoetin-delta (sold as forexample Dynepo), epoetin-omega (sold as for example Epomax), epoetinzeta (sold as for example Silapo and Reacrit) as well as for exampleEpocept, EPOTrust, Erypro Safe, Repoeitin, Vintor, Epofit, Erykine,Wepox, Espogen, Relipoeitin, Shanpoietin, Zyrop and EPIAO). In oneembodiment, the CDK4/6 inhibitor combination is administered prior toadministration of the hematopoietic growth factor. In one embodiment,the hematopoietic growth factor administration is timed so that theCDK4/6 inhibitor combination's effect on HSPCs has dissipated. In oneembodiment, the growth factor is administered at least 20 hours afterthe administration of a CDK4/6 inhibitor combination described herein.

If desired, multiple doses of a CDK4/6 inhibitor combination describedherein can be administered to the subject. Alternatively, the subjectcan be given a single dose of a CDK4/6 inhibitor combination describedherein.

In one embodiment, the activity of an active compound for a purposedescribed herein can be augmented through conjugation to an agent thattargets the diseased or abnormally proliferating cell or otherwiseenhances activity, delivery, pharmacokinetics or other beneficialproperty.

For example, the CDK4/6 inhibitor or other therapeutic agent the CDK4/6inhibitor is combined with during treatment, or combined in a treatmentregimen, can be administered as an antibody-drug conjugate (ADC). Incertain embodiments, one or more of the combination agents describedherein can be administered in conjugation or combination with anantibody or antibody fragment. Fragments of an antibody can be producedthrough chemical or genetic mechanisms. The antibody fragment can be anantigen binding fragment. For example, the antigen binding fragment canbe selected from an Fab, Fab′, (Fab′)2, or Fv. The antibody fragment canbe a Fab. Monovalent F(ab) fragments have one antigen binding site. Theantibody can be a divalent (Fab′)2 fragment, which has two antigenbinding regions that are linked by disulfide bonds. In one embodiment,the antigen fragment is a (Fab′). Reduction of F(ab′)2 fragmentsproduces two monovalent Fab′ fragments, which have a free sulfhydrylgroup that is useful for conjugation to other molecules.

A selected compound described herein can be administered in conjugationor combination with a Fv fragment. Fv fragments are the smallestfragment made from enzymatic cleavage of IgG and IgM class antibodies.Fv fragments have the antigen-binding site made of the VH and VCregions, but they lack the CH1 and CL regions. The VH and VL chains areheld together in Fv fragments by non-covalent interactions.

In one embodiment, a selected compound as described herein can beadministered in combination with an antibody fragment selected from thegroup consisting of an ScFv, domain antibody, diabody, triabody,tetrabody, Bis-scFv, minibody, Fab2, or Fab3 antibody fragment. In oneembodiment, the antibody fragment is a ScFv. Genetic engineering methodsallow the production of single chain variable fragments (ScFv), whichare Fv type fragments that include the VH and VL domains linked with aflexible peptide. When the linker is at least 12 residues long, the ScFvfragments are primarily monomeric. Manipulation of the orientation ofthe V-domains and the linker length creates different forms of Fvmolecules. Linkers that are 3-11 residues long yield scFv molecules thatare unable to fold into a functional Fv domain. These molecules canassociate with a second scFv molecule, to create a bivalent diabody. Inone embodiment, the antibody fragment administered in combination with aselected compound described herein is a bivalent diabody. If the linkerlength is less than three residues, scFv molecules associate intotriabodies or tetrabodies. In one embodiment, the antibody fragment is atriabody. In one embodiment, the antibody fragment is a tetrabody.Multivalent scFvs possess greater functional binding affinity to theirtarget antigens than their monovalent counterparts by having binding totwo more target antigens, which reduces the off-rate of the antibodyfragment. In one embodiment, the antibody fragment is a minibody.Minibodies are scFv-CH3 fusion proteins that assemble into bivalentdimers. In one embodiment, the antibody fragment is a Bis-scFv fragment.Bis-scFv fragments are bispecific. Miniaturized ScFv fragments can begenerated that have two different variable domains, allowing theseBis-scFv molecules to concurrently bind to two different epitopes.

In one embodiment, a selected compound described herein is administeredin conjugation or combination with a bispecific dimer (Fab2) ortrispecific dimer (Fab3). Genetic methods are also used to createbispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab3). Theseantibody fragments are able to bind 2 (Fab2) or 3 (Fab3) differentantigens at once.

In one embodiment, a selected compound described herein is administeredin conjugation or combination with an rIgG antibody fragment. rIgGantibody fragments refers to reduced IgG (75,000 daltons) or half-IgG.It is the product of selectively reducing just the hinge-regiondisulfide bonds. Although several disulfide bonds occur in IgG, those inthe hinge-region are most accessible and easiest to reduce, especiallywith mild reducing agents like 2-mercaptoethylamine (2-MEA). Half-IgGare frequently prepared for the purpose of targeting the exposinghinge-region sulfhydryl groups that can be targeted for conjugation,either antibody immobilization or enzyme labeling.

In other embodiments, a selected active compound described herein can belinked to a radioisotope to increase efficacy, using methods well knownin the art. Any radioisotope that is useful against Rb-positive cancercells can be incorporated into the conjugate, for example, but notlimited to, ¹³¹I, ¹²³I, ¹⁹²Ir, ³²P, ⁹⁰Sr, ¹⁹⁸Au, ²²⁶Ra, ⁹⁰Y, ²⁴¹Am,²⁵²Cf, ⁶⁰Co, or ¹³⁷Cs.

Of note, the linker chemistry can be important to efficacy andtolerability of the drug conjugates. The thio-ether linked T-DM1increases the serum stability relative to a disulfide linker version andappears to undergo endosomal degradation, resulting in intra-cellularrelease of the cytotoxic agent, thereby improving efficacy andtolerability, See, Barginear, M. F. and Budman, D. R., Trastuzumab-DM1:A review of the novel immune-conjugate for HER2-overexpressing breastcancer, The Open Breast Cancer Journal, 1:25-30, 2009.

Examples of early and recent antibody-drug conjugates, discussing drugs,linker chemistries and classes of targets for product development thatmay be used in the present invention can be found in the reviews byCasi, G. and Neri, D., Antibody-drug conjugates: basic concepts,examples and future perspectives, J. Control Release 161(2):422-428,2012, Chari, R. V., Targeted cancer therapy: conferring specificity tocytotoxic drugs, Acc. Chem. Rev., 41(1):98-107, 2008, Sapra, P. andShor, B., Monoclonal antibody-based therapies in cancer: advances andchallenges, Pharmacol. Ther., 138(3):452-69, 2013, Schliemann, C. andNeri, D., Antibody-based targeting of the tumor vasculature, Biochim.Biophys. Acta., 1776(2):175-92, 2007, Sun, Y., Yu, F., and Sun, B. W.,Antibody-drug conjugates as targeted cancer therapeutics, Yao Xue XueBao, 44(9):943-52, 2009, Teicher, B. A., and Chari, R. V., Antibodyconjugate therapeutics: challenges and potential, Clin. Cancer Res.,17(20):6389-97, 2011, Firer, M. A., and Gellerman, G. J., Targeted drugdelivery for cancer therapy: the other side of antibodies, J. Hematol.Oncol., 5:70, 2012, Vlachakis, D. and Kossida, S., Antibody DrugConjugate bioinformatics: drug delivery through the letterbox, Comput.Math. Methods Med., 2013; 2013:282398, Epub 2013 Jun. 19, Lambert, J.M., Drug-conjugated antibodies for the treatment of cancer, Br. J. Clin.Pharmacol., 76(2):248-62, 2013, Concalves, A., Tredan, O., Villanueva,C. and Dumontet, C., Antibody-drug conjugates in oncology: from theconcept to trastuzumab emtansine (T-DM1), Bull. Cancer,99(12):1183-1191, 2012, Newland, A. M., Brentuximab vedotin: aCD-30-directed antibody-cytotoxic drug conjugate, Pharmacotherapy,33(1):93-104, 2013, Lopus, M., Antibody-DM1 conjugates as cancertherapeutics, Cancer Lett., 307(2):113-118, 2011, Chu, Y. W. and Poison,A., Antibody-drug conjugates for the treatment of B-cell non-Hodgkin'slymphoma and leukemia, Future Oncol., 9(3):355-368, 2013, Bertholjotti,I., Antibody-drug conjugate—a new age for personalized cancer treatment,Chimia, 65(9): 746-748, 2011, Vincent, K. J., and Zurini, M., Currentstrategies in antibody engineering: Fc engineering and pH-dependentantigen binding, bispecific antibodies and antibody drug conjugates,Biotechnol. J., 7(12):1444-1450, 2012, Haeuw, J. F., Caussanel, V., andBeck, A., Immunoconjugates, drug-armed antibodies to fight againstcancer, Med. Sci., 25(12):1046-1052, 2009 and Govindan, S. V., andGoldenberg, D. M., Designing immunoconjugates for cancer therapy, ExpertOpin. Biol. Ther., 12(7):873-890, 2012.

Pharmaceutical Compositions and Dosage Forms

An active compound described herein, or its salt, isotopic analog, orprodrug can be administered in combination in an effective amount to thehost using any suitable approach which achieves the desired therapeuticresult. The amount and timing of active compound administered will, ofcourse, be dependent on the host being treated, the instructions of thesupervising medical specialist, on the time course of the exposure, onthe manner of administration, on the pharmacokinetic properties of theparticular active compound, and on the judgment of the prescribingphysician. Thus, because of host to host variability, the dosages givenbelow are a guideline and the physician can titrate doses of thecompound to achieve the treatment that the physician considersappropriate for the host. In considering the degree of treatmentdesired, the physician can balance a variety of factors such as age andweight of the host, presence of preexisting disease, as well as presenceof other diseases. Pharmaceutical formulations can be prepared for anydesired route of administration including, but not limited to, oral,intravenous, or aerosol administration, as discussed in greater detailbelow.

The therapeutically effective dosage of any active compound describedherein will be determined by the health care practitioner depending onthe condition, size and age of the patient as well as the route ofdelivery. In one non-limited embodiment, a dosage from about 0.1 toabout 200 mg/kg has therapeutic efficacy, with all weights beingcalculated based upon the weight of the active compound, including thecases where a salt is employed. In some embodiments, the dosage can bethe amount of compound needed to provide a serum concentration of theactive compound of up to between about 1 and 5, 10, 20, 30, or 40 μM. Insome embodiments, a dosage from about 10 mg/kg to about 50 mg/kg isemployed for oral administration. A dosage from about 0.5 mg/kg to 5mg/kg can be employed for intramuscular injection. In some embodiments,dosages can be from about 1 μmol/kg to about 50 μmol/kg, or, optionally,between about 22 μmol/kg and about 33 μmol/kg of the compound forintravenous or oral administration. An oral dosage form can include anyappropriate amount of active material, including for example from 5 mgto, 50, 100, 200, 250, 300, 400 or 500 mg per tablet or other soliddosage form.

In accordance with the presently disclosed methods, pharmaceuticallyactive compounds as described herein can be administered orally as asolid or as a liquid, or can be administered intramuscularly,intravenously, or by inhalation as a solution, suspension, or emulsion.In some embodiments, the compounds or salts also can be administered byinhalation, intravenously, or intramuscularly as a liposomal suspension.When administered through inhalation the active compound or salt can bein the form of a plurality of solid particles or droplets having anydesired particle size, and for example, from about 0.01, 0.1 or 0.5 toabout 5, 10, 20 or more microns, and optionally from about 1 to about 2microns. Compounds as disclosed in the present invention havedemonstrated good pharmacokinetic and pharmacodynamics properties, forinstance when administered by the oral or intravenous routes.

The pharmaceutical formulations can comprise an active compounddescribed herein or a pharmaceutically acceptable salt thereof, in anypharmaceutically acceptable carrier. If a solution is desired, water maybe the carrier of choice for water-soluble compounds or salts. Withrespect to the water-soluble compounds or salts, an organic vehicle,such as glycerol, propylene glycol, polyethylene glycol, or mixturesthereof, can be suitable. In the latter instance, the organic vehiclecan contain a substantial amount of water. The solution in eitherinstance can then be sterilized in a suitable manner known to those inthe art, and for illustration by filtration through a 0.22-micronfilter. Subsequent to sterilization, the solution can be dispensed intoappropriate receptacles, such as depyrogenated glass vials. Thedispensing is optionally done by an aseptic method. Sterilized closurescan then be placed on the vials and, if desired, the vial contents canbe lyophilized.

In addition to the active compounds or their salts, the pharmaceuticalformulations can contain other additives, such as pH-adjustingadditives. In particular, useful pH-adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the formulations can contain antimicrobialpreservatives. Useful antimicrobial preservatives include methylparaben,propylparaben, and benzyl alcohol. An antimicrobial preservative istypically employed when the formulations is placed in a vial designedfor multi-dose use. The pharmaceutical formulations described herein canbe lyophilized using techniques well known in the art.

For oral administration a pharmaceutical composition can take the formof solutions, suspensions, tablets, pills, capsules, powders, and thelike. Tablets containing various excipients such as sodium citrate,calcium carbonate and calcium phosphate may be employed along withvarious disintegrants such as starch (e.g., potato or tapioca starch)and certain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate,and talc are often very useful for tableting purposes. Solidcompositions of a similar type may be employed as fillers in soft andhard-filled gelatin capsules. Materials in this connection also includelactose or milk sugar as well as high molecular weight polyethyleneglycols. When aqueous suspensions and/or elixirs are desired for oraladministration, the compounds of the presently disclosed host matter canbe combined with various sweetening agents, flavoring agents, coloringagents, emulsifying agents and/or suspending agents, as well as suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof.

In yet another embodiment, there are provided injectable, stable,sterile formulations comprising combinations of active compound asdescribed herein, or a salt thereof, in a unit dosage form in a sealedcontainer. The combination is provided in the form of a lyophilizate,which is capable of being reconstituted with a suitable pharmaceuticallyacceptable carrier to form liquid formulation suitable for injectionthereof into a host. When the combination is substantiallywater-insoluble, a sufficient amount of emulsifying agent, which isphysiologically acceptable, can be employed in sufficient quantity toemulsify the compound or salt in an aqueous carrier. Particularly usefulemulsifying agents include phosphatidyl cholines and lecithin.

Additional embodiments provided herein include liposomal formulations ofthe active compounds disclosed herein. The technology for formingliposomal suspensions is well known in the art. When the compound is anaqueous-soluble salt, using conventional liposome technology, the samecan be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the active compound, the active compound can besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed can be of any conventionalcomposition and can either contain cholesterol or can becholesterol-free. When the active compound of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced can be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations comprising the active compounds disclosedherein can be lyophilized to produce a lyophilizate, which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

Pharmaceutical formulations also are provided which are suitable foradministration as an aerosol by inhalation. These formulations comprisea solution or suspension of a desired compound or combination describedherein or a salt thereof, or a plurality of solid particles of thecompound or salt or combination. The desired formulations can be placedin a small chamber and nebulized. Nebulization can be accomplished bycompressed air or by ultrasonic energy to form a plurality of liquiddroplets or solid particles comprising the compounds or salts orcombinations. The liquid droplets or solid particles may for examplehave a particle size in the range of about 0.5 to about 10 microns, andoptionally from about 0.5 to about 5 microns. In one embodiment, thesolid particles provide for controlled release through the use of adegradable polymer. The solid particles can be obtained by processingthe solid compound or a salt thereof, in any appropriate manner known inthe art, such as by micronization. Optionally, the size of the solidparticles or droplets can be from about 1 to about 2 microns. In thisrespect, commercial nebulizers are available to achieve this purpose.The compounds can be administered via an aerosol suspension ofrespirable particles in a manner set forth in U.S. Pat. No. 5,628,984,the disclosure of which is incorporated herein by reference in itsentirety.

Pharmaceutical formulations also are provided which provide a controlledrelease of a one or both compounds of the combinations described herein,including through the use of a degradable polymer, as known in the art.

When the pharmaceutical formulations suitable for administration as anaerosol is in the form of a liquid, the formulations can comprise awater-soluble active compound in a carrier that comprises water. Asurfactant can be present, which lowers the surface tension of theformulations sufficiently to result in the formation of droplets withinthe desired size range when hosted to nebulization.

The term “pharmaceutically acceptable salts” as used herein refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with hosts (e.g., human hosts) without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe presently disclosed host matter.

Thus, the term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of the presently disclosed compounds. Thesesalts can be prepared during the final isolation and purification of thecompounds or by separately reacting the purified compound in its freebase form with a suitable organic or inorganic acid and isolating thesalt thus formed. Basic compounds are capable of forming a wide varietyof different salts with various inorganic and organic acids. Acidaddition salts of the basic compounds are prepared by contacting thefree base form with a sufficient amount of the desired acid to producethe salt in the conventional manner. The free base form can beregenerated by contacting the salt form with a base and isolating thefree base in the conventional manner. The free base forms may differfrom their respective salt forms in certain physical properties such assolubility in polar solvents. Pharmaceutically acceptable base additionsalts may be formed with metals or amines, such as alkali and alkalineearth metal hydroxides, or of organic amines. Examples of metals used ascations, include, but are not limited to, sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines include, but are notlimited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, N-methylglucamine, and procaine. Thebase addition salts of acidic compounds are prepared by contacting thefree acid form with a sufficient amount of the desired base to producethe salt in the conventional manner. The free acid form can beregenerated by contacting the salt form with an acid and isolating thefree acid in a conventional manner. The free acid forms may differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents.

Salts can be prepared from inorganic acids sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric,phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, laurylsulphonate and isethionate salts,and the like. Salts can also be prepared from organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. and the like. Representativesalts include acetate, propionate, caprylate, isobutyrate, oxalate,malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Pharmaceuticallyacceptable salts can include cations based on the alkali and alkalineearth metals, such as sodium, lithium, potassium, calcium, magnesium andthe like, as well as non-toxic ammonium, quaternary ammonium, and aminecations including, but not limited to, ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. Also contemplated are the saltsof amino acids such as arginate, gluconate, galacturonate, and the like.See, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19, which isincorporated herein by reference.

Rb-Positive Cancers and Proliferative Disorders

In particular, the CDK4/6 inhibitor combinations and methods describedherein can be used to treat a subject suffering from an Rb-positivecancer or other Rb-positive abnormal cellular proliferative disorder. Insome embodiments, the cancer or cellular proliferation disorder is aCDK4/6-replication dependent cancer or cellular proliferation disorder,which refers to a cancer or cellular proliferation disorder thatrequires the activity of CDK4/6 for replication or proliferation, orwhich may be growth inhibited through the activity of a selective CDK4/6inhibitor. Cancers and disorders of such type can be characterized by(e.g., that has cells that exhibit) the presence of a functionalRetinoblastoma protein. Such cancers and disorders are classified asbeing Rb-positive. Rb-positive abnormal cellular proliferationdisorders, and variations of this term as used herein, refer todisorders or diseases caused by uncontrolled or abnormal cellulardivision which are characterized by the presence of a functionalRetinoblastoma protein, which can include cancers. In one aspect of thepresent invention, the use of CDK4/6 inhibitors in combination withadditional therapeutic agents and methods described herein can be usedto treat a non-cancerous Rb-positive abnormal cellular proliferationdisorder. Examples of such disorders may include non-malignantlymphoproliferation, non-malignant breast neoplasms, psoriasis,arthritis, dermatitis, pre-cancerous colon lesions or pulps,angiogenesis disorders, immune mediated and non-immune mediatedinflammatory diseases, arthritis, age-related macular degeneration,diabetes, and other non-cancerous or benign cellular proliferationdisorders.

Targeted cancers suitable for administration of a compound describedherein may include Rb-positive: estrogen-receptor positive cancer,HER2-negative advanced breast cancer, late-line metastatic breastcancer, liposarcoma, non-small cell lung cancer, liver cancer, ovariancancer, glioblastoma, refractory solid tumors, retinoblastoma positivebreast cancer as well as retinoblastoma positive endometrial, vaginaland ovarian cancers and lung and bronchial cancers, adenocarcinoma ofthe colon, adenocarcinoma of the rectum, central nervous system germcell tumors, teratomas, estrogen receptor-negative breast cancer,estrogen receptor-positive breast cancer, familial testicular germ celltumors, HER2-negative breast cancer, HER2-positive breast cancer, malebreast cancer, ovarian immature teratomas, ovarian mature teratoma,ovarian monodermal and highly specialized teratomas, progesteronereceptor-negative breast cancer, progesterone receptor-positive breastcancer, recurrent breast cancer, recurrent colon cancer, recurrentextragonadal germ cell tumors, recurrent extragonadal non-seminomatousgerm cell tumor, recurrent extragonadal seminomas, recurrent malignanttesticular germ cell tumors, recurrent melanomas, recurrent ovarian germcell tumors, recurrent rectal cancer, stage III extragonadalnon-seminomatous germ cell tumors, stage III extragonadal seminomas,stage III malignant testicular germ cell tumors, stage III ovarian germcell tumors, stage IV breast cancers, stage IV colon cancers, stage IVextragonadal non-seminomatous germ cell tumors, stage IV extragonadalseminoma, stage IV melanomas, stage IV ovarian germ cell tumors, stageIV rectal cancers, testicular immature teratomas, testicular matureteratomas. In particular embodiments, the targeted cancers includedestrogen-receptor positive, HER2-negative advanced breast cancer,late-line metastatic breast cancer, liposarcoma, non-small cell lungcancer, liver cancer, ovarian cancer, glioblastoma, refractory solidtumors, retinoblastoma positive breast cancer as well as retinoblastomapositive endometrial, vaginal and ovarian cancers and lung and bronchialcancers, metastatic colorectal cancer, metastatic melanoma with CDK4mutation or amplification, or cisplatin-refractory, unresectable germcell tumors.

In one embodiment, the Rb-positive cancer is selected from anRb-positive carcinoma, sarcoma, including, but not limited to, lungcancer, bone cancer, pancreatic cancer, skin cancer, cancer of the heador neck, cutaneous or intraocular melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer, coloncancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,prostate cancer, cancer of the bladder, cancer of the kidney or ureter,renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of thecentral nervous system (CNS), primary CNS lymphoma, spinal axis tumors,brain stem glioma, pituitary adenoma, or a combination of one or more ofthe foregoing cancers.

In one embodiment, the Rb-positive cancer is selected from the groupconsisting of Rb-positive: fibrosarcoma, myxosarcoma, chondrosarcoma,osteosarcoma, chordoma, malignant fibrous histiocytoma, hemangiosarcoma,angiosarcoma, lymphangiosarcoma. Mesothelioma, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma; epidermoid carcinoma,malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellularcarcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma,transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cellcarcinoma, glioma anaplastic; glioblastoma multiforme, neuroblastoma,medulloblastoma, malignant meningioma, malignant schwannoma,neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma ofthyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma,malignant carcinoid, malignant paraganglioma, melanoma, Merkel cellneoplasm, cystosarcoma phylloide, salivary cancers, thymic carcinomas,bladder cancer, and Wilms tumor.

In more particular embodiments, the Rb-positive cancer or disorderincludes a blood disorder or a hematologic malignancy, including, butnot limited to, myeloid disorder, lymphoid disorder, leukemia, lymphoma,myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), mastcell disorder, and myeloma (e.g., multiple myeloma), among others.Abnormal proliferation of T-cells, B-cells, and/or NK-cells can resultin a wide range of diseases such as cancer, proliferative disorders andinflammatory/immune diseases. A host, for example a human, afflictedwith any of these disorders can be treated with an effective amount of acombination as described herein to achieve a decrease in symptoms (apalliative agent) or a decrease in the underlying disease (a diseasemodifying agent).

Examples include T-cell or NK-cell lymphoma, for example, but notlimited to: peripheral T-cell lymphoma; anaplastic large cell lymphoma,for example anaplastic lymphoma kinase (ALK) positive, ALK negativeanaplastic large cell lymphoma, or primary cutaneous anaplastic largecell lymphoma; angioimmunoblastic lymphoma; cutaneous T-cell lymphoma,for example mycosis fungoides, Sezary syndrome, primary cutaneousanaplastic large cell lymphoma, primary cutaneous CD30+ T-celllymphoproliferative disorder; primary cutaneous aggressiveepidermotropic CD8+ cytotoxic T-cell lymphoma; primary cutaneousgamma-delta T-cell lymphoma; primary cutaneous small/medium CD4+ T-celllymphoma, and lymphomatoid papulosis; Adult T-cell Leukemia/Lymphoma(ATLL); Blastic NK-cell Lymphoma; Enteropathy-type T-cell lymphoma;Hematosplenic gamma-delta T-cell Lymphoma; Lymphoblastic Lymphoma; NasalNK/T-cell Lymphomas; Treatment-related T-cell lymphomas; for examplelymphomas that appear after solid organ or bone marrow transplantation;T-cell prolymphocytic leukemia; T-cell large granular lymphocyticleukemia; Chronic lymphoproliferative disorder of NK-cells; AggressiveNK cell leukemia; Systemic EBV+ T-cell lymphoproliferative disease ofchildhood (associated with chronic active EBV infection); Hydroavacciniforme-like lymphoma; Adult T-cell leukemia/lymphoma;Enteropathy-associated T-cell lymphoma; Hepatosplenic T-cell lymphoma;or Subcutaneous panniculitis-like T-cell lymphoma.

In one embodiment, a combination disclosed herein can be used in aneffective amount to treat a host, for example a human, with a lymphomaor lymphocytic or myelocytic proliferation disorder or abnormality. Forexample, the compounds as described herein can be administered to a hostsuffering from a Hodgkin Lymphoma or a Non-Hodgkin Lymphoma. Forexample, the host can be suffering from a Non-Hodgkin Lymphoma such as,but not limited to: an AIDS-Related Lymphoma; Anaplastic Large-CellLymphoma; Angioimmunoblastic Lymphoma; Blastic NK-Cell Lymphoma;Burkitt's Lymphoma; Burkitt-like Lymphoma (Small Non-Cleaved CellLymphoma); Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma;Cutaneous T-Cell Lymphoma; Diffuse Large B-Cell Lymphoma;Enteropathy-Type T-Cell Lymphoma; Follicular Lymphoma; HepatosplenicGamma-Delta T-Cell Lymphoma; Lymphoblastic Lymphoma; Mantle CellLymphoma; Marginal Zone Lymphoma; Nasal T-Cell Lymphoma; PediatricLymphoma; Peripheral T-Cell Lymphomas; Primary Central Nervous SystemLymphoma; T-Cell Leukemias; Transformed Lymphomas; Treatment-RelatedT-Cell Lymphomas; or Waldenstrom's Macroglobulinemia.

Alternatively, a combination disclosed herein, or its salt, prodrug, orisotopic variant can be used in an effective amount to treat a host, forexample a human, with a Hodgkin Lymphoma, such as, but not limited to:Nodular Sclerosis Classical Hodgkin's Lymphoma (CHL); Mixed CellularityCHL; Lymphocyte-depletion CHL; Lymphocyte-rich CHL; LymphocytePredominant Hodgkin Lymphoma; or Nodular Lymphocyte Predominant HL.

Alternatively, a combination disclosed herein, or its salt, prodrug, orisotopic variant can be used in an effective amount to treat a host, forexample a human with a specific B-cell lymphoma or proliferativedisorder such as, but not limited to: multiple myeloma; Diffuse large Bcell lymphoma; Follicular lymphoma; Mucosa-Associated Lymphatic Tissuelymphoma (MALT); Small cell lymphocytic lymphoma; Mediastinal large Bcell lymphoma; Nodal marginal zone B cell lymphoma (NMZL); Splenicmarginal zone lymphoma (SMZL); Intravascular large B-cell lymphoma;Primary effusion lymphoma; or Lymphomatoid granulomatosis; B-cellprolymphocytic leukemia; Hairy cell leukemia; Splenic lymphoma/leukemia,unclassifiable; Splenic diffuse red pulp small B-cell lymphoma; Hairycell leukemia-variant; Lymphoplasmacytic lymphoma; Heavy chain diseases,for example, Alpha heavy chain disease, Gamma heavy chain disease, Muheavy chain disease; Plasma cell myeloma; Solitary plasmacytoma of bone;Extraosseous plasmacytoma; Primary cutaneous follicle center lymphoma; Tcell/histiocyte rich large B-cell lymphoma; DLBCL associated withchronic inflammation; Epstein-Barr virus (EBV)+ DLBCL of the elderly;Primary mediastinal (thymic) large B-cell lymphoma; Primary cutaneousDLBCL, leg type; ALK+ large B-cell lymphoma; Plasmablastic lymphoma;Large B-cell lymphoma arising in HHV8-associated multicentric; Castlemandisease; B-cell lymphoma, unclassifiable, with features intermediatebetween diffuse large B-cell lymphoma; or B-cell lymphoma,unclassifiable, with features intermediate between diffuse large B-celllymphoma and classical Hodgkin lymphoma.

In one embodiment, a combination disclosed herein, or its salt, prodrug,or isotopic variant can be used in an effective amount to treat a host,for example a human with leukemia. For example, the host may besuffering from an acute or chronic leukemia of a lymphocytic ormyelogenous origin, such as, but not limited to: Acute lymphoblasticleukemia (ALL); Acute myelogenous leukemia (AML); Chronic lymphocyticleukemia (CLL); Chronic myelogenous leukemia (CML); juvenilemyelomonocytic leukemia (JMML); hairy cell leukemia (HCL); acutepromyelocytic leukemia (a subtype of AML); large granular lymphocyticleukemia; or Adult T-cell chronic leukemia. In one embodiment, thepatient suffers from an acute myelogenous leukemia, for example anundifferentiated AML (M0); myeloblastic leukemia (M1; with/withoutminimal cell maturation); myeloblastic leukemia (M2; with cellmaturation); promyelocytic leukemia (M3 or M3 variant [M3V]);myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]);monocytic leukemia (M5); erythroleukemia (M6); or megakaryoblasticleukemia (M7).

The presence or normal functioning of the retinoblastoma (Rb) tumorsuppressor protein (Rb-positive) can be determined through any of thestandard assays known to one of ordinary skill in the art, including butnot limited to Western Blot, ELISA (enzyme linked immunoadsorbentassay), IHC (immunohistochemistry), and FACS (fluorescent activated cellsorting). The selection of the assay will depend upon the tissue, cellline or surrogate tissue sample that is utilized e.g., for exampleWestern Blot and ELISA may be used with any or all types of tissues,cell lines or surrogate tissues, whereas the IHC method would be moreappropriate wherein the tissue utilized in the methods of the presentinvention was a tumor biopsy. FACs analysis would be most applicable tosamples that were single cell suspensions such as cell lines andisolated peripheral blood mononuclear cells. See for example, US20070212736 “Functional Immunohistochemical Cell Cycle Analysis as aPrognostic Indicator for Cancer”. Alternatively, molecular genetictesting may be used for determination of retinoblastoma gene status.Molecular genetic testing for retinoblastoma includes the following asdescribed in Lohmann and Gallie “Retinoblastoma. Gene Reviews” (2010):“A comprehensive, sensitive and economical approach for the detection ofmutations in the RB1 gene in retinoblastoma” Journal of Genetics, 88(4),517-527 (2009).

In some embodiments, the cancer to be treated is selected fromestrogen-receptor positive, HER2-negative advanced breast cancer,late-line metastatic breast cancer, liposarcoma, non-small cell lungcancer, liver cancer, ovarian cancer, glioblastoma, refractory solidtumors, retinoblastoma positive breast cancer as well as retinoblastomapositive endometrial, vaginal and ovarian cancers and lung and bronchialcancers.

Dosing Regimen

In one aspect of the invention, dosing regimens are provided wherein ahost suffering from an Rb-positive cellular proliferative disorder isadministered a CDK4/6 inhibitor described herein to arrest the cells inG0 or G1 phase, thus synchronizing the cells, wherein upon dissipationof the cell cycle arrest caused by the CDK4/6 inhibitor, the host isadministered a second chemotherapeutic agent that takes advantage of thecells reentry into the cell cycle so that the administered second agentis more effective against a larger number of cells, as more cells willbe in the effective cell-cycle state that the second chemotherapeutic ismost efficacious in, for example, within the G1, S, G2, or M phase. Thusin one embodiment, the invention includes administering in combinationor alternation a CDK4/6 inhibitor described herein, for example a CDK4/6inhibitor selected from Table 1, in an effective amount to a hostsuffering from an Rb-positive abnormal cellular proliferation disorderin a treatment regimen, wherein (either alone or in any combinationthereof, each of which is considered specifically and independentlydescribed): (i) a substantial portion of the abnormal cells (e.g., atleast 80% or greater) are arrested in G0 or G1 and re-enter the cellcycle in less than about 24 hours, 30 hours, 36 hours, or 48 hours fromthe last administration of a CDK4/6 inhibitor described herein; (ii) asubstantial portion of the abnormal cells reenter the cell-cyclesynchronously in less than about 24 hours, 30 hours, 36 hours, or 48hours from the last administration of the CDK4/6 inhibitor describedherein; (iii) the dissipation of the CDK4/6 inhibitor's inhibitoryeffect on the abnormal cells occurs in less than about 24 hours, 30hours, 36 hours, or 48 hours from the administration of the CDK4/6inhibitor; (iv) a substantial portion of the abnormal cells reenter thecell-cycle in less than abou t24 hours, 30 hours, 36 hours, or 48 hoursfrom the dissipation of the CDK4/6 inhibitor's inhibitory effect; or(vi) a substantial portion of the abnormal cells reenter the cell-cyclewithin less than about 24 hours, about 30 hours, about 36 hours, orabout 48 hours from the point in which the administered CDK4/6 inhibitorcompound's concentration level in the subject's blood drops below atherapeutic effective concentration, and, either just prior to,concomitantly with, or shortly after the re-entry of the abnormal cellsas described in (i)-(vi), administering at least one additionalchemotherapeutic agent.

For example, a CDK4/6 inhibitor compound can be administered so thatCDK4/6-replication dependent abnormal cells are G1 arrested as theyproceed through the cell cycle, wherein, due to the dissipation of theG1-arresting effect of the CDK4/6 inhibitor, a significant number ofcells reenter the cell-cycle in a synchronous manner and are capable ofreplicating shortly after exposure, for example, within about 24-48hours or less, and continue to replicate as they are exposed to thesecond chemotherapeutic agent. In one embodiment, the CDK4/6 inhibitorcompound is administered to allow for the cycling of theCDK4/6-replication dependent abnormal cells between G1-arrest andreentry into the cell-cycle to accommodate a repeated-dosing treatmentregimen, for example a long term repeated-dosing treatment regime.

By synchronizing the abnormal cells, more cells can be exposed to anagent that acts at a particular cell-cycle phase, allowing for doseintensification (e.g., more therapy can be given in a fixed period oftime) and fewer dosing requirements, which will translate to betterefficacy. Therefore, the presently disclosed methods can result inregimens that are less toxic and more effective. When appropriate, thesmall molecules can be formulated for oral, topical, intranasal,inhalation, intravenous or any other desired form of administration.

A compound useful in the methods described herein is a selective CDK4/6inhibitor compound that selectively inhibit at least one of CDK4 andCDK6 or through the inhibition of cellular replication of an Rb-positivecancer. In one embodiment, the compounds described herein have an IC₅₀for CDK4 as measured in a CDK4/CycD1 IC₅₀ phosphorylation assay that isat least 500 times or greater lower than the compound's IC₅₀s for CDK2as measured in a CDK2/CycE IC₅₀ phosphorylation assay.

The use of a compound as described herein can induce selective G1 arrestin CDK4/6-dependent abnormal cells such as Rb-positive abnormal cells(e.g., as measured in a cell-based in vitro assay). In one embodiment,the CDK4/6 inhibitor is capable of increasing the percentage ofCDK4/6-dependent abnormal cells in the G1 phase, while decreasing thepercentage of CDK4/6-dependent abnormal cells in the G2/M phase and Sphase. In one embodiment, the compound induces substantially pure (i.e.,“clean”) G1 cell cycle arrest in the CDK4/6-dependent abnormal cells,e.g., wherein treatment with the CDK4/6 inhibitor compound induces cellcycle arrest such that the majority of abnormal cells are arrested in G1as defined by standard methods (e.g. propidium iodide (PI) staining orothers) with the population of cells in the G2/M and S phases combinedbeing less than about 30%, about 25%, about 20%, about 15%, about 10%,about 5%, about 3% or less of the total cell population. Methods ofassessing the cell phase of a population of cells are known in the art(see, for example, in U.S. Patent Application Publication No.2002/0224522) and include cytometric analysis, microscopic analysis,gradient centrifugation, elutriation, fluorescence techniques includingimmunofluorescence, and combinations thereof. Cytometric techniquesinclude exposing the cell to a labeling agent or stain, such asDNA-binding dyes, e.g., PI, and analyzing cellular DNA content by flowcytometry. Immunofluorescence techniques include detection of specificcell cycle indicators such as, for example, thymidine analogs (e.g.,5-bromo-2-deoxyuridine (BrdU) or an iododeoxyuridine), with fluorescentantibodies.

In certain embodiments, the compound administered to induce G1 arrest isselected from the group consisting of the compound or a compositioncomprising Formula I, Formula II, Formula III, Formula IV, or Formula V,or a pharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof. In certain embodiments, the compound administered isselected from a compound contained in Table 1, or a pharmaceuticallyacceptable composition, salt, isotopic analog, or prodrug thereof.

T-Cell Neoplasm Dosing Regimen

In another aspect of the present invention, provided herein is adifferential dosing method for treating certain malignancies. Forexample, in one aspect of the invention, provided herein is a method oftreating an Rb-positive T-cell malignancy by administering a CDK4/6inhibitor described herein at a dose that provides for the extendedinhibition of the proliferation of the T-cell malignancy, but providesfor the differential proliferation of non-diseased Rb-positivehematopoietic cells, for example, hematopoietic stem and progenitorcells (HSPCs).

It has been discovered that T-cell lineages are particularly sensitiveto the CDK4/6 inhibitors described herein. Accordingly, provided hereinis a method for treating patients with particular T-cell malignancieswith a concentration of a CDK4/6 inhibitor which inhibits theproliferation of the T-cell malignancy, but differentially inhibits, forexample, inhibits for a shorter period or at a lesser rate, otherhematopoietic lineage cells. Because of this, the use of a CDK4/6inhibitor to treat T-cell malignancies allows for an extended period oftreatment while reducing the side effects, such as myelosuppression,associated with long term use of CDK4/6 inhibitors.

In one embodiment, the dose administered to the host having a T-cellmalignancy in a dose effective enough to inhibit the abnormal T-cellproliferation while differentially inhibiting other hematopoietic celllineages so that the other cell lineages are allowed to re-enter thecell cycle at a faster rate than the T-cell malignancy, allowing for thecontinued replication of hematopoetic cells. In one embodiment of theinvention, provided herein is a method of treating a host suffering froma T-cell malignancy comprising 1) administering to the host a CDK4/6inhibitor in an amount sufficient to inhibit the proliferation of thespecific T-cell malignancy, 2) measuring or analyzing the cell cyclestatus of the specific cell T-cell malignancy and at least one subset ofnon-diseased hematopoietic cell lineages, for example, long termhematopoietic stem cells (LT-HSCs), short term hematopoietic stem cells(ST-HSCs), hematopoietic progenitor cells (HPCs), multipotentprogenitors (MPPs), common myeloid progenitors (CMPs), common lymphoidprogenitors (CLPs), granulocyte-monocyte progenitors (GMPs), andmegakaryocyte-erythroid progenitors (MEPs), macrophage progenitors,granulocyte progenitors (GP), monocyte progenitors (MP), megakaryocyteprogenitors (MP), erythroid progenitors (EP), B cell progenitors, T celland NK cell progenitors (TNKP), T cell progenitors (TCP), NK cellprogenitors (TNKP), mast cell progenitors, macrophage-dendritic cellprogenitors, dendritic cell progenitors, osteoclast precursor cells,basophil progenitors, eosinophil pregenitors, neutrophil progenitors,oligodendrocyte pre-progenitors (OPPs), or a combination thereof, 3)adjusting the dose of the CDK4/6 inhibitor so that at least one subsetof non-diseased hematopoietic cell lineages, for example, long termhematopoietic stem cells (LT-HSCs), short term hematopoietic stem cells(ST-HSCs), hematopoietic progenitor cells (HPCs), multipotentprogenitors (MPPs), common myeloid progenitors (CMPs), common lymphoidprogenitors (CLPs), granulocyte-monocyte progenitors (GMPs), andmegakaryocyte-erythroid progenitors (MEPs), macrophage progenitors,granulocyte progenitors (GP), monocyte progenitors (MP), megakaryocyteprogenitors (MP), erythroid progenitors (EP), B cell progenitors, T celland NK cell progenitors (TNKP), T cell progenitors (TCP), NK cellprogenitors (TNKP), mast cell progenitors, macrophage-dendritic cellprogenitors, dendritic cell progenitors, osteoclast precursor cells,basophil progenitors, eosinophil pregenitors, neutrophil progenitors,oligodendrocyte pre-progenitors (OPPs), or a combination thereof,reenter the cell cycle while a significant portion of the T-cellmalignant cells remain in cell cycle arrest. In a particular embodiment,the host is a human, and the non-diseased hematological lineage cellsare selected from HSC/MPPs (CD45dim/CD34+/CD38−), OPPs(CD45dim/CD34+/CD38+), monocyte progenitors (CD45+/CD14+/CD11b+),granulocyte progenitors (CD45+/CD14−/CD11b+), erythroid progenitors(CD45−/CD71+), and megakaryocyte progenitors (CD45+/CD61+). Themeasurement of cell cycle status of cell lineages is described furtherbelow and routine in the art. Cell lineages are readily assayable byanalyzing specific cell markers on their cell surface: see Stelzer etal., CD45 gating for routine flow cytometric analysis of human bonemarrow specimens, Annals of New York Academy of Sciences, Mar. 20, 1993,Vol. 677; pg. 265-280; Spangrude et al., Purification andCharacterization of Mouse Hematopoietic Stem Cells, Science (1988) Vol.241:58-62.

Thus, as contemplated herein, provided is a method of treating a T-celldisorder comprising administering a CDK4/6 inhibitor at a dose whereinabnormal T-cells, or a significant portion of abnormal T-cells, forexample 50%, 60%, 70%, 80% or greater, are arrested in the G0 and/or G1phase of the cell cycle while at least a significant number, for example50%, 60%, 70%, 80% or greater, of one non-diseased hematopoietic celllineage is not arrested at the G0 and/or G1 phase of the cell cycle.

Hematological Malignances Differential Dosing

It has been discovered that hematological malignancies respond to CDK4/6inhibitors in a differential fashion. Accordingly, this differentialresponse provides for the differential dosing of a CDK4/6 inhibitorbased on the specific hematological disorder the host may be sufferingfrom. Thus, in one aspect of the invention, provided herein is a methodof treating a host suffering from a hematological cellular proliferationdisorder comprising 1) identifying the specific hematologicaldeficiency, 2) administering to the host a CDK4/6 inhibitor in an amountsufficient to inhibit the proliferation of a significant portion of, forexample, at least 50%, at least 60%, at least 70%, at least 80%, thespecific hematological deficiency, 3) measuring, or analyzing theresults of a measurement of, the cell cycle status of the specifichematological deficient cells as well as one or more subsets ofnon-diseased hematological cell lineages, 4) adjusting the dose of theCDK4/6 inhibitor to the minimal amount necessary so that a significantportion of the diseased cells remain in G0 and/or G1 cell cycle arrest,while a significant portion, for example, at least 50%, at least 60%, atleast 70%, at least 80%, or at least one subset of non-diseasedhematological cells are mpt om G0 and/or G1 cell cycle arrest.Determining the cell cycle status of specific hematological celllineages is well known in the art and described further in the Examplesbelow.

Optimizing Dosage and Time Regimens Using CDK4/6 Inhibitor forChemoprotection

As indicated in FIGS. 12-19 , cells of the bone marrow aredifferentially affected by the CDK4/6 inhibitors described herein. Thisdifferential response should be taken into account when treatingpatients through the selection of optimal dosage and timing. Forexample, if a patient has depleted macrophages due to the administrationof a DNA-damaging chemotherapeutic agent for the treatment of anRb-negative cancer, for example, it may be appropriate to use a CDK4/6inhibitor as a chemoprotectant for some hematopoietic cell lineageswhile sparing the macrophage progenitor cells the inhibition caused bythe use of the CDK4/6 inhibitor. For example, as FIG. 14 indicates,macrophage lineages are less sensitive to CDK4/6 inhibition than otherhematological cell lines. Accordingly, a method for differential dosingthat takes into account the sparing of this cell lineage when needed,while still protecting other cell lines from the damage associated withthe chemotherapeutic is provided.

Preparation of Active Compounds Syntheses

The disclosed compounds can be made by the following general schemes:

In Scheme 1, Ref-1 is WO 2010/020675 A1; Ref-2 is White, J. D.; et al.J. Org. Chem. 1995, 60, 3600; and Ref-3 Presser, A. and Hufner, A.Monatshefte für Chemie 2004, 135, 1015.

In Scheme 2, Ref-1 is WO 2010/020675 A1; Ref-4 is WO 2005/040166 A1; andRef-5 is Schoenauer, K and Zbiral, E. Tetrahedron Letters 1983, 24, 573.

In Scheme 3, Ref-1 is WO 2010/020675 A1.

In Scheme 8, Ref-1 is WO 2010/020675 A1; Ref-2 is WO 2005/040166 A1; andRef-3 is Schoenauer, K and Zbiral, E. Tetrahedron Letters 1983, 24, 573.

Alternatively, the lactam can be generated by reacting the carboxylicacid with a protected amine in the presence of a strong acid and adehydrating agent, which can be together in one moiety as a strong acidanhydride. Examples of strong acid anhydrides include, but are notlimited to, trifluoroacetic acid anhydride, tribromoacetic acidanhydride, trichloroacetic acid anhydride, or mixed anhydrides. Thedehydrating agent can be a carbodiimide based compound such as but notlimited to DCC (N,N-dicyclohexylcarbodiimide), EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or DIC(N,N-diisopropylcarbodiimide). An additional step may be necessary totake off the N-protecting group and the methodologies are known to thoseskilled in the art.

Alternatively, the halogen moiety bonded to the pyrimidine ring can besubstituted with any leaving group that can be displaced by a primaryamine, for example to create an intermediate for a final product such asBr, I, F, SMe, SO₂Me, SOalkyl, SO₂alkyl. See, for ExamplePCT/US2013/037878 to Tavares.

Other amine intermediates and final amine compounds can be synthesizedby those skilled in the art. It will be appreciated that the chemistrycan employ reagents that comprise reactive functionalities that can beprotected and de-protected and will be known to those skilled in the artat the time of the invention. See for example, Greene, T. W. and Wuts,P. G. M., Greene's Protective Groups in Organic Synthesis, 4^(th)edition, John Wiley and Sons.

CDK4/6 Inhibitors of the present invention can be synthesized accordingto the generalized Scheme 9. Specific synthesis and characterization ofthe Substituted 2-aminopyrmidines can be found in, for instance,WO2012/061156.

Compounds T, Q, GG, and U were prepared as above and were characterizedby mass spectrometry and NMR as shown below:

Compound T

1H NMR (600 MHz, DMSO-d6) □ ppm 1.47 (br. s., 6H) 1.72 (br. s., 2H) 1.92(br. s., 2H) 2.77 (br. s., 3H) 3.18 (br. s., 2H) 3.46 (br. s., 2H) 3.63(br. s., 2H) 3.66 (d, J=6.15 Hz, 2H) 3.80 (br. s., 2H) 7.25 (s, 1H) 7.63(br. s., 2H) 7.94 (br. s., 1H) 8.10 (br. s., 1H) 8.39 (br. s., 1H) 9.08(br. s., 1H) 11.59 (br. s., 1H). LCMS ESI (M+H) 447.

Compound Q

1H NMR (600 MHz, DMSO-d6) □ ppm 0.82 (d, J=7.32 Hz, 2H) 1.08-1.37 (m,3H) 1.38-1.64 (m, 2H) 1.71 (br. s., 1H) 1.91 (br. s., 1H) 2.80 (br. s.,1H) 3.12 (s, 1H) 3.41 (br. s., 4H) 3.65 (br. s., 4H) 4.09 (br. s., 1H)7.26 (s, 1H) 7.52-7.74 (m, 2H) 7.94 (br. s., 1H) 8.13 (br. s., 1H) 8.40(br. s., 1H) 9.09 (br. s., 1H) 9.62 (br. s., 1H) 11.71 (br. s., 1H).LCMS ESI (M+H) 433.

Compound GG

1H NMR (600 MHz, DMSO-d6) □ ppm 0.85 (br. s., 1H) 1.17-1.39 (m, 7H)1.42-1.58 (m, 2H) 1.67-1.84 (m, 3H) 1.88-2.02 (m, 1H) 2.76-2.93 (m, 1H)3.07-3.22 (m, 1H) 3.29-3.39 (m, 1H) 3.41-3.61 (m, 4H) 3.62-3.76 (m, 4H)3.78-3.88 (m, 1H) 4.12 (br. s., 1H) 7.28 (s, 1H) 7.60-7.76 (m, 2H) 7.98(s, 1H) 8.13 (br. s., 1H) 8.41 (s, 1H) 9.10 (br. s., 1H) 11.21 (br. s.,1H) 11.54 (s, 1H). LCMS ESI (M+H) 475.

Compound U

1H NMR (600 MHz, DMSO-d6) □ ppm 0.84 (t, J=7.61 Hz, 2H) 1.13-1.39 (m,4H) 1.46 (d, J=14.05 Hz, 2H) 1.64-1.99 (m, 6H) 2.21 (br. s., 1H)2.66-2.89 (m, 2H) 3.06 (br. s., 1H) 3.24-3.36 (m, 1H) 3.37-3.50 (m, 2H)3.56-3.72 (m, 2H) 3.77-4.00 (m, 4H) 4.02-4.19 (m, 2H) 7.25 (s, 1H)7.50-7.75 (m, 2H) 7.89 (d, J=2.93 Hz, 1H) 8.14 (d, J=7.32 Hz, 1H) 8.38(br. s., 1H) 9.06 (s, 1H) 11.53 (br. s., 1H). LCMS ESI (M+H) 517.

EXAMPLES

Intermediates B, E, K, L, 1A, 1F and 1CA were synthesized according toU.S. Pat. No. 8,598,186 entitled CDK Inhibitors to Tavares, F. X. andStrum, J. C.

The patents WO 2013/148748 entitled Lactam Kinase Inhibitors to Tavares,F. X., WO 2013/163239 entitled Synthesis of Lactams to Tavares, F. X.,and U.S. Pat. No. 8,598,186 entitled CDK Inhibitors to Tavares, F. X.and Strum, J. C. are incorporated by reference herein in their entirety.

Example 1 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4yl)amino]ethyl]carbamate, Compound 1

To a solution of 5-bromo-2,4-dichloropyrimidine (3.2 g, 0.0135 mol) inethanol (80 mL) was added Hunig's base (3.0 mL) followed by the additionof a solution of N-(tert-butoxycarbonyl)-1,2-diaminoethane (2.5 g,0.0156 mole) in ethanol (20 mL). The contents were stirred overnight for20 hrs. The solvent was evaporated under vacuum. Ethyl acetate (200 mL)and water (100 mL) were added and the layers separated. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum. Column chromatography on silica gel using hexane/ethyl acetate(0-60%) afforded tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. ¹HNMR(d6-DMSO) δ ppm 8.21 (s, 1H), 7.62 (brs, 1H), 7.27 (brs, 1H), 3.39 (m,2H), 3.12 (m, 2H), 1.34 (s, 9H). LCMS (ESI) 351 (M+H).

Example 2 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate,Compound 2

To tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate (1.265 g,3.6 mmol) in THE (10 mL) was added the acetal (0.778 mL, 5.43 mmol),Pd(dppf)CH₂Cl₂ (148 mg), and triethylamine (0.757 mL, 5.43 mmol). Thecontents were degassed and then purged with nitrogen. To this was thenadded CuI (29 mg). The reaction mixture was heated at reflux for 48 hrs.After cooling, the contents were filtered over CELITE™ and concentrated.Column chromatography of the resulting residue using hexane/ethylacetate (0-30%) afforded tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.¹H NMR (d6-DMSO) δ ppm 8.18 (s, 1H), 7.63 (brs, 1H), 7.40 (brs, 1H),5.55 (s, 1H), 3.70 (m, 2H), 3.60 (m, 2H), 3.42 (m, 2H), 3.15 (m, 2H),1.19-1.16 (m, 15H). LCMS (ESI) 399 (M+H).

Example 3 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 3

To a solution of the coupled product (2.1 g, 0.00526 mole) in THE (30mL) was added TBAF solid (7.0 g). The contents were heated to andmaintained at 65 degrees for 2 hrs. Concentration followed by columnchromatography using ethyl acetate/hexane (0-50%) afforded tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale brown liquid (1.1 g). ¹HNMR (d6-DMSO) δ ppm 8.88 (s, 1H), 6.95(brs, 1H), 6.69 (s, 1H), 5.79 (s, 1H), 4.29 (m, 2H), 3.59 (m, 4H), 3.34(m, 1H), 3.18 (m, 1H), 1.19 (m, 9H), 1.17 (m, 6H). LCMS (ESI) 399 (M+H).

Example 4 Synthesis of tert-butylN-[2-(2-chloro-6-formyl-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]carbamate,Compound 4

To the acetal (900 mg) from the preceeding step was added AcOH (8.0 mL)and water (1.0 mL). The reaction was stirred at room temperature for 16hrs. Conc. and column chromatography over silica gel using ethylacetate/hexanes (0-60%) afforded tert-butylN-[2-(2-chloro-6-formyl-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]carbamate asa foam (0.510 g). ¹HNMR (d6-DMSO) δ ppm 9.98 (s, 1H), 9.18 (s, 1H), 7.66(s, 1H), 6.80 (brs, 1H), 4.52 (m, 2H), 4.36 (m, 2H), 1.14 (s, 9H). LCMS(ESI) 325 (M+H).

Example 5 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 5

To the aldehyde (0.940 g) from the preceeding step in DMF (4 mL) wasadded oxone (1.95 g, 1.1 eq). The contents were stirred at room temp for7 hrs. Silica gel column chromatography using hexane/ethyl acetate(0-100%) afforded7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.545 g). ¹HNMR (d6-DMSO) δ ppm 9.11 (s, 1H), 7.39 (s, 1H), 4.38(m, 2H), 4.15 (m, 2H), 1.48 (m, 9H). LCMS (ESI) 341 (M+H).

Example 6 Synthesis of methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylate,Compound 6

To a solution of 2-chloro-7-propyl-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.545 g, 0.00156 mole) from the preceeding step in toluene (3.5mL) and MeOH (1 mL) was added TMS-diazomethane (1.2 mL). After stirringovernight at room temperature, the excess of TMS-diazomethane wasquenched with acetic acid (3 mL) and the reaction was concentrated undervacuum. The residue was purified by silica gel column chromatographywith hexane/ethyl acetate (0-70%) to afford methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylateas an off white solid (0.52 g). ¹HNMR (d6-DMSO) δ ppm 9.10 (s, 1H), 7.45(s, 1H), 6.81 (brs, 1H) 4.60 (m, 2H), 3.91 (s, 3H), 3.29 (m, 2H), 1.18(m, 9H) LCMS (ESI) 355 (M+H).

Example 7 Synthesis of Chloro Tricyclic Amide, Compound 7

To methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylate(0.50 g, 0.0014 mole) from the preceeding step in dichloromethane (2.0mL) was added TFA (0.830 mL). The contents were stirred at roomtemperature for 1 hr. Concentration under vacuum afforded the crudeamino ester which was suspended in toluene (5 mL) and Hunig's base (0.5mL). The contents were heated at reflux for 2 hrs. Concentrationfollowed by silica gel column chromatography using hexane/ethyl acetate(0-50%) afforded the desired chloro tricyclic amide (0.260 g). ¹HNMR(d6-DMSO) δ ppm 9.08 (s, 1H), 8.48 (brs, 1H), 7.21 (s, 1H) 4.33 (m, 2H),3.64 (m, 2H). LCMS (ESI) 223 (M+H).

Example 8 Synthesis of chloro-N-methyltricyclic amide, Compound 8

To a solution of the chloro tricycliclactam, Compound 7, (185 mg,0.00083 mole) in DMF (2.0 mL) was added sodium hydride (55% dispersionin oil, 52 mg). After stirring for 15 mins, methyl iodide (62 μL, 1.2eq). The contents were stirred at room temperature for 30 mins. Afterthe addition of methanol (5 mL), sat NaHCO₃ was added followed by theaddition of ethyl acetate. Separation of the organic layer followed bydrying with magnesium sulfate and concentration under vacuum affordedthe N-methylated amide in quantitative yield. ¹HNMR (d6-DMSO) δ ppm 9.05(s, 1H), 7.17 (s, 1H) 4.38 (m, 2H), 3.80 (m, 2H), 3.05 (s, 3H). LCMS(ESI) 237 (M+H).

Example 9 Synthesis of 1-methyl-4-(6-nitro-3-pyridyl)piperazine,Compound 9

To 5-bromo-2-nitropyridine (4.93 g, 24.3 mmole) in DMF (20 mL) was addedN-methylpiperazine (2.96 g, 1.1 eq) followed by the addition of DIPEA(4.65 mL, 26.7 mmole). The contents were heated at 90 degrees for 24hrs. After addition of ethyl acetate (200 mL), water (100 mL) was addedand the layers separated. Drying followed by concentration afforded thecrude product which was purified by silica gel column chromatographyusing (0-10%) DCM/Methanol. ¹HNMR (d6-DMSO) δ ppm 8.26 (s, 1H), 8.15(1H, d, J=9.3 Hz), 7.49 (1H, d, J=9.4 Hz), 3.50 (m, 4H), 2.49 (m, 4H),2.22 (s, 3H).

Example 10 Synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine,Compound 10

To 1-methyl-4-(6-nitro-3-pyridyl)piperazine (3.4 g) in ethyl acetate(100 mL) and ethanol (100 mL) was added 10% Pd/C (400 mg) and then thereaction was stirred under hydrogen (10 psi) overnight. After filtrationthrough CELITE™, the solvents were evaporated and the crude product waspurified by silica gel column chromatography using DCM/7N ammonia inMeOH (0-5%) to afford 5-(4-methylpiperazin-1-yl)pyridin-2-amine (2.2 g).¹HNMR (d6-DMSO) δ ppm 7.56 (1H, d, J=3 Hz), 7.13 (1H, m), 6.36 (1H, d,J=8.8 Hz), 5.33 (brs, 2H), 2.88 (m, 4H), 2.47 (m, 4H), 2.16 (s, 3H).

Example 11 Synthesis of tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate, Compound 11

This compound was prepared as described in WO 2010/020675 A1.

Example 12 Synthesis of tert-butylN-[2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, Compound 12

To benzyl N-[1-(hydroxymethyl)-2-methyl-propyl]carbamate (11.0 g, 0.0464mole) in dioxane (100 mL) cooled to 0° C. was added diphenylphosphorylazide (10.99 mL, 1.1 eq) followed by the addition of DBU (8.32 mL, 1.2eq). The contents were allowed to warm to room temperature and stirredfor 16 hrs. After the addition of ethyl acetate (300 mL) and water (100mL), the organic layer was separated and washed with satd. NaHCO₃ (100mL). The organic layer was then dried (magnesium sulfate) andconcentrated under vacuum. To this intermediate in DMSO (100 mL) wasadded sodium azide (7.54 g) and the contents then heated to 90 degreesfor 2 hrs. After addition of ethyl acetate and water the layers wereseparated. The organic layer was dried with magnesium sulfate followedby concentration under vacuum to afford an oil that was purified bysilica gel column chromatography using hexane/ethyl acetate (0-70%) toafford benzyl N-[1-(azidomethyl)-2-methyl-propyl] carbamate 6.9 g as acolorless oil.

To benzyl N-[1-(azidomethyl)-2-methyl-propyl] carbamate (6.9 g, 0.0263mole) in THE (100 mL) was added triphenyl phosphine (7.59 g, 1.1 eq).The contents were stirred for 20 hrs. After addition of water (10 mL),and stirring for an additional 6 hrs, ethyl acetate was added and thelayers separated. After drying with magnesium sulfate and concentrationunder vacuum, the crude product was purified by silica gel columnchromatography using DCM/MeOH (0-10%) to afford benzylN-[1-(aminomethyl)-2-methyl-propyl] carbamate as a yellow oil.

To benzyl N-[1-(aminomethyl)-2-methyl-propyl] carbamate (4.65 g, 0.019mole) in THE (70 mL) was added 2N NaOH (20 mL) followed by the additionof di-tert-butyl dicarbonate (5.15 g, 1.2 eq). After stirring for 16hrs, ethyl acetate was added and the layers separated. After drying withmagnesium sulfate and concentration under vacuum, the crude product waspurified using hexane/ethyl acetate (0-40%) over a silica gel column toafford intermediate A, tert-butylN-[2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, (6.1 g). ¹HNMR(600 MHz, CHLOROFORM-d) δ ppm 0.89 (d, J=6.73 Hz, 3H) 0.92 (d, J=6.73Hz, 3H) 1.38 (s, 9H) 1.70-1.81 (m, 1H) 3.18 (d, J=5.56 Hz, 2H) 3.47-3.60(m, 1H) 4.76 (s, 1H) 4.89 (d, J=7.90 Hz, 1H) 5.07 (s, 2H) 7.25-7.36 (m,5H). LCMS (ESI) 337 (M+H).

Example 13 Synthesis of tert-butylN-[2-(benzyloxycarbonylamino)-4-methyl-pentyl] carbamate, Compound 13

To a solution of benzyl N-[1-(hydroxymethyl)-3-methyl-butyl]carbamate(6.3 g, 0.025 mole) in DCM (100 mL) was added diisopropylethyl amine(5.25 mL, 1.2 eq) followed by the addition of methane sulfonylchloride(2.13 mL, 1.1 eq) at 0 degrees. After stirring for 3 hrs, water (100 mL)was added and the organic layer separated. After drying with magnesiumsulfate and concentration under vacuum, the crude[2-(benzyloxycarbonylamino)-4-methyl-pentyl]methanesulfonate which wastaken directly to the next step.

To the crude [2-(benzyloxycarbonylamino)-4-methyl-pentyl]methanesulfonate from the above reaction in DMF (50 mL), was addedsodium azide 2.43 g. The reaction mixture was then heated to 85 degreesfor 3 hrs. After cooling, ethyl acetate (300 mL) and water was added.The organic layer was separated, dried with magnesium sulfate and thenconcentrated under vacuum to afford the crude benzylN-[1-(azidomethyl)-3-methyl-butyl] carbamate. To this crude intermediatewas added THE (100 mL) followed by triphenylphosphine 7.21 g and stirredunder nitrogen for 16 hrs. After addition of water (10 mL), and stirringfor an additional 6 hrs, ethyl acetate was added and the layersseparated. After drying with magnesium sulfate and concentration undervacuum, the crude product was columned using DCM/MeOH (0-10%) to affordbenzyl N-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g).

To benzyl N-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g, 0.018mole) in THE (60 mL) was added 2N NaOH (18 mL) followed by the additionof di-tert-butyl dicarbonate (4.19 g, 1.07 eq). After stirring for 16hrs, ethyl acetate was added and the layers separated. After drying withmagnesium sulfate and concentration under vacuum, the crude product wastaken to the next step. ¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.89 (d,J=6.73 Hz, 6H) 1.25-1.34 (m, 1H) 1.39 (s, 9H) 1.57-1.71 (m, 2H)3.04-3.26 (m, 2H) 3.68-3.80 (m, 1H) 4.72-4.89 (m, 2H) 5.06 (s, 2H)7.25-7.38 (m, 5H). LCMS (ESI) 351 (M+H).

Example 14 Synthesis of tert-butylN-[(2R)-2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, Compound14

Compound 14 was synthesized from benzylN-[(1R)-1-(hydroxymethyl)-2-methyl-propyl]carbamate using similarsynthetic steps as that described for Compound 13. The analytical data(NMR and mass spec) was consistent with that for Compound 12.

Example 15 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3-methyl-butyl]carbamate, Compound 15

Compound 15 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2-methyl-propyl]carbamate using similarsynthetic steps as that described for Compound 13. The analytical data(NMR and mass spec) was consistent with that for Compound 12.

Example 16 Synthesis of tert-butylN-[(1S)-1-(aminomethyl)-2-methyl-propyl]carbamate, Compound 16

To a solution of tert-butylN-[(1S)-1-(hydroxymethyl)-2-methyl-propyl]carbamate carbamate (6.3 g,0.025 mole) in THE (100 mL) was added diisopropylethyl amine (5.25 mL,1.2 eq) followed by the addition of methane sulfonylchloride (2.13 mL,1.1 eq) at 0 degrees. After stirring for 3 hrs, water (100 mL) was addedand the organic layer separated. After drying with magnesium sulfate andconcentration under vacuum, the crude[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl] methanesulfonate wastaken directly to the next step.

To the crude [(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]methanesulfonate from the above reaction in DMSO (50 mL), was addedsodium azide (2.43 g). The reaction mixture was then heated to 85degrees for 3 hrs. After cooling, ethyl acetate (300 mL) and water wereadded. The organic layer was separated, dried with magnesium sulfate andthen concentrated under vacuum to afford the crude benzylN-[1-(azidomethyl)-3-methyl-butyl] carbamate. To this crude intermediatewas added THE (100 mL) followed by triphenylphosphine (7.21 g) and thereaction was stirred under nitrogen for 16 hrs. After addition of water(10 mL), and stirring for an additional 6 hrs, ethyl acetate was addedand the layers separated. After drying with magnesium sulfate andconcentration under vacuum, the crude product was purified by silica gelcolumn chromatography using DCM/MeOH (0-10%) to afford benzylN-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g). LCMS (ESI) 203(M+H).

Example 17 Synthesis of tert-butylN-[(1R)-1-(aminomethyl)-2-methyl-propyl]carbamate, Compound 17

Compound 17 was synthesized from tert-butylN-[(1R)-1-(hydroxymethyl)-2-methyl-propyl] carbamate using a similarsynthetic sequence as described for Compound 16. The analytical data(NMR and mass spec) was consistent with Compound 16.

Example 18 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-4-methyl-pentyl] carbamate, Compound18

Compound 18 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-3-methyl-butyl]carbamate using a similarsynthetic sequence as described for Compound 13. The analytical data(NMR and mass spec) was consistent with Compound 13.

Example 19 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-2-phenyl-ethyl] carbamate, Compound19

Compound 19 was synthesized from benzylN-[(1S)-2-hydroxy-1-phenyl-ethyl] carbamate using a similar syntheticsequence as described for Compound 13. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.20-1.33 (m, 9H) 3.11 (t, J=6.29 Hz, 2H) 4.59-4.68 (m, 1H) 4.88-5.01(m, 2H) 6.81 (t, J=5.42 Hz, 1H) 7.14-7.35 (m, 10H) 7.69 (d, J=8.49 Hz,1H). LCMS (ESI) 371 (M+H).

Example 20 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3-methyl-pentyl] carbamate, Compound20

Compound 20 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2-methyl-butyl]carbamate using a similarsynthetic sequence as described for Compound 13. ¹HNMR (600 MHz,CHLOROFORM-d) δ ppm 0.85-0.92 (m, 6H) 1.05-1.15 (m, 1H) 1.35-1.41 (m,9H) 1.45-1.56 (m, 2H) 3.14-3.24 (m, 2H) 3.54-3.64 (m, 1H) 4.78 (s, 1H)4.96 (d, J=7.91 Hz, 1H) 5.06 (s, 2H) 7.27-7.37 (m, 5H). LCMS (ESI) 351(M+H).

Example 21 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3,3-dimethyl-butyl] carbamate,Compound 21

Compound 21 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2,2-dimethyl-propyl]carbamate using a similarsynthetic sequence as described for Compound 13. LCMS (ESI) 351.

Example 22 Synthesis of tert-butylN-[[1-(benzyloxycarbonylamino)cyclohexyl]methyl] carbamate, Compound 22

To a solution of benzyl N-[1-(aminomethyl)cyclohexyl]carbamate (10.0 g,0.0381 mole) in THE (150 mL) was added di-tert-butyl dicarbonate (9.15g, 1.1 eq) and the contents were stirred at room temperature for 16 hrs.Ethyl acetate and water were then added. The organic layer wasseparated, dried over magnesium sulfate and then concentrated undervacuum to afford tert-butylN-[[1-(benzyloxycarbonylamino)cyclohexyl]methyl] carbamate (13.1 g).¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.92-1.54 (m, 17H) 1.76-2.06 (m, 2H) 3.09(d, J=6.15 Hz, 2H) 4.92 (s, 2H) 6.63 (d, J=17.27 Hz, 1H) 7.16-7.49 (m,6H). LCMS (ESI) 363 (M+H).

Example 23 Synthesis of tert-butylN-[[1-(benzyloxycarbonylamino)cyclopentyl]methyl] carbamate, Compound 23

tert-butyl N-[[1-(benzyloxycarbonylamino)cyclopentyl]methyl]carbamatewas synthesized in an analogous manner to tert-butylN-[[1-(benzyloxycarbonylamino) cyclohexyl]methyl] carbamate. LCMS (ESI)349 (M+H).

Example 24 Synthesis of 2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine,Compound 24

To 5-bromo-2-nitropyridine (1.2 g, 5.9 mmol) in DMSO (4 mL) was added1-(4-piperidyl)piperidine (1.0 g, 5.9 mmole) and triethylamine (0.99 mL,7.1 mmole). The contents were heated to 120° C. in a CEM Discoverymicrowave system for 3 hours. The crude reaction was then purified bysilica gel column chromatography with DCM/methanol (0-20%) to afford2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine as an oil (457 mg).¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.26-1.36 (m, 2H) 1.43 (m, 6H) 1.76 (m,2H) 2.37 (m, 5H) 2.94 (t, J=12.74 Hz, 2H) 4.06 (d, J=13.47 Hz, 2H) 7.41(dd, J=9.37, 2.64 Hz, 1H) 8.08 (d, J=9.37 Hz, 1H) 8.20 (d, J=2.64 Hz,1H).

Example 25 Synthesis of 5-[4-(1-piperidyl)-1-piperidyl]pyridin-2-amine,Compound 25

5-[4-(1-piperidyl)-1-piperidyl]pyridin-2-amine was prepared in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.13-1.37 (m, 6H) 1.40-1.63 (m, 6H) 1.71 (m, 2H), 2.24 (m, 1H) 2.43(m, 2H) 3.33 (d, J=12.30 Hz, 2H) 5.31 (s, 2H) 6.33 (d, J=8.78 Hz, 1H)7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.64 Hz, 1H). LCMS (ESI) 261(M+H).

Example 26 Synthesis of 4-[1-(6-nitro-3-pyridyl)-4-piperidyl]morpholine, Compound 26

4-[1-(6-nitro-3-pyridyl)-4-piperidyl]morpholine was synthesized in amanner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.41 (m, 2H) 1.82 (m, 2H) 2.42 (m, 5H) 2.98 (t, J=12.44Hz, 2H) 3.52 (s, 4H) 4.04 (d, J=12.88 Hz, 2H) 7.42 (d, J=9.37 Hz, 1H)8.08 (d, J=9.08 Hz, 1H) 8.21 (s, 1H).

Example 27 Synthesis of 5-(4-morpholino-1-piperidyl) pyridin-2-amine,Compound 27

5-(4-morpholino-1-piperidyl)pyridin-2-amine was prepared in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.34-1.52 (m, 2H) 1.78 (m, 2H) 2.14 (m, 1H) 2.43 (m, 4H) 3.32 (d,J=12.30 Hz, 4H) 3.47-3.59 (m, 4H) 5.32 (s, 2H) 6.34 (d, J=8.78 Hz, 1H)7.11 (dd, J=8.93, 2.78 Hz, 1H) 7.47-7.62 (m, 1H). LCMS (ESI) 263 (M+H).

Example 28 Synthesis of 4-[1-(6-nitro-3-pyridyl)-4-piperidyl]thiomorpholine, Compound 28

4-[1-(6-nitro-3-pyridyl)-4-piperidyl] thiomorpholine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.40-1.52 (m, 2H) 1.71 (m, 2H) 2.49-2.55 (m, 4H)2.56-2.63 (m, 1H) 2.68-2.75 (m, 4H) 2.88-2.98 (m, 2H) 4.09 (d, J=13.18Hz, 2H) 7.42 (dd, J=9.22, 3.07 Hz, 1H) 8.08 (d, J=9.37 Hz, 1H) 8.20 (d,J=3.22 Hz, 1H).

Example 29 Synthesis of 5-(4-thiomorpholino-1-piperidyl)pyridin-2-amine, Compound 29

5-(4-thiomorpholino-1-piperidyl) pyridin-2-amine was prepared in amanner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.47-1.59 (m, 2H) 1.65 (m, 2H) 2.22-2.38 (m, 1H) 2.50-2.59 (m, 6H)2.68-2.82 (m, 4H) 3.33 (d, J=12.00 Hz, 2H) 5.31 (s, 2H) 6.33 (d, J=9.08Hz, 1H) 7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.64 Hz, 1H). LCMS(ESI) 279 (M+H).

Example 30 Synthesis of 2-nitro-5-(1-piperidyl)pyridine, Compound 30

2-nitro-5-(1-piperidyl) pyridine was synthesized in a manner similar tothat used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.56 (m, 6H) 3.49 (d, J=4.39 Hz, 4H) 7.30-7.47 (m, 1H)8.02-8.12 (m, 1H) 8.15-8.26 (m, 1H).

Example 31 Synthesis of 5-(1-piperidyl)pyridin-2-amine, Compound 31

5-(1-piperidyl) pyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.39-1.46 (m, 2H) 1.51-1.62 (m, 4H)2.75-2.92 (m, 4H) 5.30 (s, 2H) 6.34 (d, J=8.78 Hz, 1H) 7.09 (dd, J=8.78,2.93 Hz, 1H) 7.54 (d, J=2.93 Hz, 1H). LCMS (ESI) 178 (M+H).

Example 32 Synthesis of 4-(6-nitro-3-pyridyl) thiomorpholine, Compound32

4-(6-nitro-3-pyridyl) thiomorpholine was synthesized in a manner similarto that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 2.56-2.69 (m, 4H) 3.79-3.92 (m, 4H) 7.43 (dd, J=9.22,3.07 Hz, 1H) 8.10 (d, J=9.37 Hz, 1H) 8.20 (d, J=2.93 Hz, 1H).

Example 33 Synthesis of 5-thiomorpholinopyridin-2-amine, Compound 33

5-thiomorpholinopyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 2.59-2.73 (m, 4H) 3.04-3.20 (m, 4H) 5.41(s, 2H) 6.35 (d, J=8.78 Hz, 1H) 7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.57 (d,J=2.64 Hz, 1H). LCMS (ESI) 196 (M+H).

Example 34 Synthesis of tert-butyl(4R)-5-(6-nitro-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate,Compound 34

tert-butyl(4R)-5-(6-nitro-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylatewas synthesized in a manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.33 (d, J=32.21 Hz, 11H) 1.91 (m, 2H) 3.15 (d, J=10.25Hz, 1H) 3.58 (m, 1H) 4.46 (m, 1H) 4.83 (s, 1H) 7.16 (s, 1H) 7.94 (s, 1H)8.05-8.16 (m, 1H).

Example 35 Synthesis of tert-butyl(4R)-5-(6-amino-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate,Compound 35

tert-butyl(4R)-5-(6-amino-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylatewas prepared in a manner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.31 (d, J=31.91 Hz, 11H) 1.83 (m, 2H) 2.71-2.82 (m, 1H) 3.44 (m,1H) 4.30 (d, 2H) 5.08 (s, 2H) 6.35 (d, J=8.78 Hz, 1H) 6.77-6.91 (m, 1H)7.33 (s, 1H). LCMS (ESI) 291 (M+H).

Example 36 Synthesis of N,N-dimethyl-1-(6-nitro-3-pyridyl)piperidin-4-amine, Compound 36

N,N-dimethyl-1-(6-nitro-3-pyridyl)piperidin-4-amine was synthesized in amanner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.30-1.45 (m, 2H) 1.79 (m, 2H) 2.14 (s, 6H) 2.33 (m, 1H)2.92-3.04 (m, 2H) 4.03 (d, J=13.76 Hz, 2H) 7.42 (dd, J=9.22, 3.07 Hz,1H) 8.04-8.11 (m, 1H) 8.21 (d, J=2.93 Hz, 1H).

Example 37 Synthesis of 5-[4-(dimethylamino)-1-piperidyl]pyridin-2-amine, Compound 37

5-[4-(dimethylamino)-1-piperidyl]pyridin-2-amine was prepared in amanner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.35-1.50 (m, 2H) 1.69-1.81 (m, 2H) 2.00-2.10 (m, 1H) 2.11-2.22 (s,6H) 3.17-3.36 (m, 4H) 5.19-5.38 (s, 2H) 6.34 (d, J=8.78 Hz, 1H) 7.10(dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.63 Hz, 1H). LCMS (ESI) 221 (M+H).

Example 38 Synthesis of 4-(6-nitro-3-pyridyl) morpholine, Compound 38

4-(6-nitro-3-pyridyl) morpholine was synthesized in a manner similar tothat used in the synthesis of 2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine.

Example 39 Synthesis of 5-morpholinopyridin-2-amine, Compound 39

5-morpholinopyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 2.91-3.00 (m, 4H) 3.76-3.84 (m, 4H)4.19 (br. s., 2H) 6.45 (d, J=8.78 Hz, 1H) 7.12 (dd, J=8.78, 2.93 Hz, 1H)7.72 (d, J=2.93 Hz, 1H).

Example 40 Synthesis of 5-(4-isobutylpiperazin-1-yl) pyridin-2-amine,Compound 40

1-isobutyl-4-(6-nitro-3-pyridyl)piperazine was synthesized in a mannersimilar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then converted5-(4-isobutylpiperazin-1-yl)pyridin-2-amine in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.88 (d, J=6.73 Hz, 6H) 1.71-1.84(m, 1H) 2.10 (d, J=7.32 Hz, 2H) 2.46-2.58 (m, 4H) 2.97-3.07 (m, 4H) 4.12(s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.14 (dd, J=8.78, 2.93 Hz, 1H) 7.75 (d,J=2.93 Hz, 1H). LCMS (ESI) 235 (M+H).

Example 41 Synthesis of 5-(4-isopropylpiperazin-1-yl) pyridin-2-amine,Compound 41

1-isopropyl-4-(6-nitro-3-pyridyl)piperazine was synthesized in a mannersimilar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-(4-isopropylpiperazin-1-yl)pyridin-2-amine in a manner similar tothat used in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 1.06 (d, J=6.44 Hz, 6H) 2.59-2.75(m, 5H) 2.97-3.10 (m, 4H) 4.13 (s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.15 (dd,J=9.08, 2.93 Hz, 1H) 7.76 (d, J=2.93 Hz, 1H). LCMS (ESI) 221 (M+H).

Example 42 Synthesis of5-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-2-amine, Compound 42

(2S,6R)-2,6-dimethyl-4-(6-nitro-3-pyridyl)morpholine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-2-amine in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, CHLOROFORM-d)δ ppm 1.20 (d, J=6.44 Hz, 6H) 2.27-2.39 (m, 2H) 3.11-3.21 (m, 2H)3.70-3.84 (m, 2H) 4.15 (s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.12 (dd, J=8.78,2.93 Hz, 1H) 7.72 (d, J=2.63 Hz, 1H). LCMS (ESI) 208 (M+H).

Example 43 Synthesis of5-[(3R,5S)-3,5-dimethylpiperazin-1-yl]pyridin-2-amine, Compound 43

(3S,5R)-3,5-dimethyl-1-(6-nitro-3-pyridyl)piperazine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-[(3R,5S)-3,5-dimethylpiperazin-1-yl]pyridin-2-amine in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, CHLOROFORM-d)δ ppm 1.09 (d, J=6.44 Hz, 6H) 2.20 (t, J=10.83 Hz, 2H) 2.95-3.08 (m, 2H) 3.23 (dd, J=11.71, 2.05 Hz, 2H) 4.13 (s, 2H) 6.45 (d, J=8.78 Hz, 1H)7.14 (dd, J=8.78, 2.93 Hz, 1H) 7.73 (d, J=2.63 Hz, 1H). LCMS (ESI) 207(M+H).

Example 44 Synthesis of Compound 44

tert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate

A solution of intermediate A in ethanol (100 mL) was hydrogenated under30 psi of hydrogen using 10% Pd/C (0.7 g) in a pressure bomb for 7 hrs.After filtration of the reaction mixture through CELITE™, the organiclayer was concentrated under vacuum to afford tert-butylN-(2-amino-3-methyl-butyl) carbamate (3.8 g).

To a solution of 5-bromo-2,4-dichloro-pyrimidine (7.11 g, 0.0312 mole)in ethanol (100 mL) was added diisopropylethyl amine (5.45 mL, 1.0 eq)and tert-butyl N-(2-amino-3-methyl-butyl) carbamate (6.31 g, 0.0312mole). The reaction mixture was stirred at room temperature for 20 hrs.After concentration under vacuum, ethyl acetate and water were added.The organic layer was separated, dried with magnesium sulfate and thenconcentrated under vacuum. The crude product was purified by silica gelcolumn chromatography using hexane/ethyl acetate (0-30%) to affordtert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.77-0.85 (d, J=6.5 Hz, 3H)0.87 (d, J=6.73 Hz, 3H) 1.31-1.39 (m, 9H) 1.82-1.93 (m, 1H) 2.94 (d,J=5.56 Hz, 1H) 3.08-3.22 (m, 2H) 3.98 (d, J=8.20 Hz, 1H) 6.96 (d, J=8.78Hz, 1H) 8.21 (s, 1H). LCMS (ESI) 393 (M+H).

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-3-methyl-butyl]carbamate

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-3-methyl-butyl]carbamatewas synthesized by hosting tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto Sonogoshira conditions as described for tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamatefollowed by subsequent treatment with TBAF as described in the synthesisof tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.11 (d, J=6.44 Hz, 3H) 1.18 (t, J=7.03Hz, 6H) 1.21-1.26 (m, 12H) 2.88 (br. s., 1H) 3.43-3.78 (m, 6H) 3.97-4.08(m, 1H) 5.61 (s, 1H) 6.65 (s, 1H) 6.71-6.78 (m, 1H) 8.87 (s, 1H). LCMS(ESI) 441 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

To a solution tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamatein THF was added TBAF and the contents were heated at reflux for 3 hrs.Ethyl acetate and water were then added and the organic layer separated,dried with magnesium sulfate and then concentrated under vacuum. To thiscrude reaction was added acetic acid/water (9:1) and the contents werestirred for 12 hrs at room temperature. After concentration undervacuum, sat NaHCO₃ and ethyl acetate were added. The organic layer wasseparated, dried and then concentrated under vacuum. The crude reactionproduct thus obtained was dissolved in DMF, oxone was then added and thecontents stirred for 3 hrs. After addition of ethyl acetate, thereaction mixture was filtered through CELITE™ and concentrated undervacuum. Column chromatography of the crude product over silica gel usinghexane/ethyl acetate (0-100%) afforded7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=7.03 Hz, 3H) 0.97 (d,J=6.73 Hz, 3H) 1.52 (s, 9H) 1.99-2.23 (m, 1H) 3.98 (dd, J=14.05, 3.51Hz, 1H) 4.47-4.71 (m, 2H) 7.47 (s, 1H) 9.17 (s, 1H). LCMS (ESI) 383(M+H).

Compound 44

To7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.050 g, 0.00013 mole) in DCM (1.5 mL) was added DIC (32.7 mg) andDMAP (10 mg). The contents were stirred for 2 hrs. Trifluoroacetic acid(0.4 mL) was then added and stirring continued for an additional 30minutes. After addition of satd NaHCO₃ to neutralize the excess acid,ethyl acetate was added and the organic layer separated, dried usingmagnesium sulfate and then concentrated under vacuum. The crude productwas purified by silica gel column chromatography using hexane/ethylacetate (0-100%) to afford the product. ¹HNMR (600 MHz, DMSO-d₆) δ ppm0.72 (d, J=6.73 Hz, 3H) 0.97 (d, J=6.73 Hz, 3H) 2.09-2.22 (m, 1H) 3.57(dd, J=13.18, 4.98 Hz, 1H) 3.72 (dd, J=13.61, 4.25 Hz, 1H) 4.53 (dd,J=8.05, 3.95 Hz, 1H) 7.20 (s, 1H) 8.34 (d, J=4.98 Hz, 1H) 9.08 (s, 1H).LCMS (ESI) 265 (M+H).

Example 45 Synthesis of Compound 45

Compound 14 was hydrogenated with 10% Pd/C to afford the intermediatetert-butyl N-[(2R)-2-amino-3-methyl-butyl] carbamate, which was thentreated with 5-bromo-2,4-dichloro-pyrimidine using analogous reactionconditions as described for Compound 44 to afford Compound 45 Theanalytical data is consistent with that reported for the racemate(Intermediate 1A).

Example 46 Synthesis of Compound 46

Compound 15 was hydrogenated with 10% Pd/C to afford the intermediatetert-butyl N-[(2S)-2-amino-3-methyl-butyl]carbamate, which was thentreated with 5-bromo-2,4-dichloro-pyrimidine using analogous reactionconditions as described for Compound 44 to afford Compound 46. Theanalytical data (NMR and LCMS) was consistent with that reported for theracemate Compound 44.

Example 47 Synthesis of Compound 47

To a solution of Compound 44 (80 mg, 0.00030 mole) in DMF (3 mL) wasadded a 60% dispersion of sodium hydride in oil (40 mg). After stirringfor 15 minutes, methyl iodide (37 μL, 2 eq) was added. The contents werestirred at room temperature for 30 minutes. Saturated NaHCO₃ was thenadded followed by ethyl acetate. The organic layer was dried withmagnesium sulfate and then concentrated under vacuum to afford theproduct. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.74 (d, J=6.73 Hz, 3H) 0.91 (d,J=6.73 Hz, 3H) 2.04-2.20 (m, 1H) 3.04 (s, 3H) 3.69 (dd, J=13.76, 1.17Hz, 1H) 3.96 (dd, J=13.76, 4.68 Hz, 1H) 4.58 (dd, J=7.32, 3.51 Hz, 1H)7.16 (s, 1H) 9.05 (s, 1H). LCMS (ESI) 279 (M+H).

Example 48 Synthesis of Compound 48

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate

Compound 18 was hydrogenated with 10% Pd/C in ethanol under a blanket ofhydrogen at 50 psi in a pressure bomb to afford tert-butylN-[(2S)-2-amino-4-methyl-pentyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.91 (d, J=6.44 Hz, 3H) 0.94 (d,J=6.44 Hz, 3H) 1.32-1.51 (m, 11H) 1.55-1.67 (m, 1H) 3.28 (t, J=5.86 Hz,2H) 4.21-4.42 (m, 1H) 4.84 (s, 1H) 5.84 (d, J=7.32 Hz, 1H) 8.07 (s, 1H).LCMS (ESI) 407 (M+H).

To a solution of tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate(5.0 g, 12.3 mmole) in toluene (36 mL) and triethylamine (7.2 mL) wasadded under nitrogen, 3,3-diethoxyprop-1-yne (2.8 mL, 19.7 mmole),Pd₂(dba)₃ (1.1 g, 1.23 mmole), and triphenylarsine (3.8 g, 12.3 mmole).The contents were heated to 70 degrees for 24 hrs. After cooling to roomtemperature, the reaction mixture was filtered through CELITE™ and thenconcentrated under vacuum. The crude product was purified by silica gelcolumn chromatography using hexane/ethyl acetate (0-30%) to afford(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.88 (d, J=6.44 Hz, 3H) 0.97 (d,J=6.44 Hz, 3H) 1.47 (s, 9H) 1.49-1.54 (m, 1H) 1.56 (t, J=7.17 Hz, 2H)3.98 (dd, J=13.91, 3.07 Hz, 1H) 3.76 (dd, J=13.31, 4.13 Hz, 1H) 4.38 (d,J=14.05 Hz, 1H) 4.90 (t, J=7.17 Hz, 1H) 7.41 (s, 1H) 9.11 (s, 1H). LCMS(M+H) 397.

Compound 48 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d,J=6.73 Hz, 3H) 0.97 (d, J=6.44 Hz, 3H) 1.34-1.46 (m, 1H) 1.48-1.65 (m,2H) 3.40 (dd, J=13.32, 5.42 Hz, 1H) 3.76 (dd, J=13.47, 4.10 Hz, 1H)4.76-4.92 (m, 1H) 7.17 (s, 1H) 8.34 (d, J=5.27 Hz, 1H) 9.04 (s, 1H).LCMS (ESI) 279 (M+H).

Example 49 Synthesis of Compound 49

Compound 49 was synthesized in a manner similar to that described forCompound 47. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d, J=6.44 Hz, 3H) 0.97(d, J=6.44 Hz, 3H) 1.37-1.68 (m, 3H) 3.04 (s, 3H) 3.56 (d, J=13.47 Hz,1H) 4.00 (dd, J=13.32, 4.25 Hz, 1H) 4.82-4.94 (m, 1H) 7.16 (s, 1H) 9.03(s, 1H). LCMS (ESI) 293 (M+H).

Example 50 Synthesis of Compound 50

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-pentyl]carbamate

Compound 20 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-3-methyl-pentyl]carbamate which was reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-pentyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.88-0.95 (m, 6H) 1.11-1.20 (m, 1H)1.34 (s, 9H) 1.44-1.54 (m, 1H) 1.64-1.72 (m, 1H) 3.17-3.27 (m, 1H)3.33-3.43 (m, 1H) 4.11-4.21 (m, 1H) 4.81 (s, 1H) 5.92 (d, J=8.20 Hz, 1H)8.05 (s, 1H). LCMS (ESI) 407.

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3-methyl-pentyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3-methyl-pentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.76-0.89 (m, 6H) 1.03 (q, J=7.22 Hz, 3H)1.10-1.17 (m, 3H) 1.25-1.42 (m, 11H) 1.59-1.73 (m, 1H) 3.35-3.47 (m, 4H)3.51-3.73 (m, 2H) 3.99-4.11 (m, 1H) 5.52-5.56 (m, 1H) 6.76-7.03 (m, 2H)8.12-8.23 (m, 1H). LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.80 (t, J=7.47 Hz, 3H) 0.86 (d,J=7.03 Hz, 3H) 1.06-1.30 (m, 2H) 1.48 (s, 9H) 1.79-1.96 (m, 1H) 3.95(dd, J=14.05, 3.22 Hz, 1H) 4.52 (d, J=14.35 Hz, 1H) 4.61-4.73 (m, 1H)7.43 (s, 1H) 9.13 (s, 1H). LCMS (ESI) 397 (M+H).

Compound 50 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.74 (t,J=7.32 Hz, 3H) 0.89 (d, J=6.73 Hz, 3H) 1.00-1.12 (m, 2H) 1.82-1.94 (m,1H) 3.55 (dd, J=13.91, 4.83 Hz, 1H) 3.70 (dd, J=13.61, 4.25 Hz, 1H) 4.57(dd, J=7.91, 4.10 Hz, 1H) 7.17 (s, 1H) 8.31 (d, J=5.27 Hz, 1H) 9.05 (s,1H). LCMS (ESI) 279 (M+H).

Example 51 Synthesis of Compound 51

Compound 51 was synthesized in a manner similar to Compound 47. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.77 (t, J=7.47 Hz, 3H) 0.84 (d, J=6.73 Hz, 3H)1.07-1.16 (m, 2H) 1.82-1.95 (m, 1H) 3.03 (s, 3H) 3.68 (d, J=13.76 Hz,1H) 3.96 (dd, J=13.76, 4.39 Hz, 1H) 4.59-4.70 (m, 1H) 7.16 (s, 1H) 9.04(s, 1H). LCMS (ESI) 293 (M+H).

Example 52 Synthesis of Compound 52

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3,3-dimethyl-butyl]carbamate

Compound 21 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-3,3-dimethyl-butyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed using analogous reaction conditions as described fortert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3,3-dimethyl-butyl]carbamate.LCMS (ESI) 407 (M+H).

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3,3-dimethyl-butyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3,3-dimethyl-butyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2,2-dimethyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2,2-dimethyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 397 (M+H).

Intermediate 1F was synthesized using an analogous synthetic sequence asthat described for intermediate 1A. LCMS (ESI) 279 (M+H).

Example 53 Synthesis of Compound 53

Compound 53 was synthesized in a manner similar to that described forIntermediate 1CA. LCMS (ESI) 293 (M+H).

Example 54 Synthesis of Compound 54

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-phenyl-ethyl]carbamate

Compound 21 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-2-phenyl-ethyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-phenyl-ethyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.32 (s, 9H) 3.29-3.50 (m, 2H) 5.12-5.24(m, 1H) 7.10 (t, J=5.27 Hz, 1H) 7.21 (t, J=6.88 Hz, 1H) 7.26-7.34 (m,4H) 7.89 (d, J=7.32 Hz, 1H) 8.24 (s, 1H). LCMS (ESI) 427 (M+H).

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-phenyl-ethyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-phenyl-ethyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.14 (t, J=7.03 Hz, 6H) 1.32 (s, 9H) 3.39(s, 2H) 3.52-3.61 (m, 2H) 3.64-3.73 (m, 2H) 5.17-5.26 (m, 1H) 5.57 (s,1H) 7.07-7.14 (m, 1H) 7.20-7.25 (m, 1H) 7.26-7.33 (m, 4H) 7.90 (d,J=7.61 Hz, 1H) 8.19 (s, 1H). LCMS (ESI) 475 (M+H).

7-[(1S)-2-(tert-butoxycarbonylamino)-1-phenyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-2-(tert-butoxycarbonylamino)-1-phenyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 417 (M+H).

Compound 54

Compound 54 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 3.58-3.69(m, 1H) 4.13 (dd, J=13.47, 4.39 Hz, 1H) 6.07 (d, J=3.81 Hz, 1H) 6.85 (d,J=7.32 Hz, 2H) 7.19-7.31 (m, 3H) 7.34 (s, 1H) 8.27 (d, J=5.27 Hz, 1H)9.13 (s, 1H). LCMS (ESI) 299 (M+H).

Example 55 Synthesis of Compound 55

tert-butylN-[(1S)-1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]-2-methyl-propyl]carbamate

tert-butylN-[(1S)-1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]-2-methyl-propyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and Intermediate Eusing analogous reaction conditions as described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.95-1.02 (m, 6H) 1.35-1.45 (m, 9H)1.75-1.90 (m, 1H) 3.35-3.48 (m, 1H) 3.52-3.61 (m, 1H) 3.64-3.76 (m, 1H)4.56 (d, J=8.49 Hz, 1H) 6.47 (s, 1H) 8.07 (s, 1H). LCMS (ESI) 393 (M+H).

tert-butylN-[(1S)-1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]-2-methyl-propyl]carbamate

tert-butylN-[(1S)-1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]-2-methyl-propyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.90-1.00 (m, 6H) 1.18-1.25 (m, 6H)1.34-1.36 (m, 9H) 1.69-1.90 (m, 1H) 3.34-3.82 (m, 6H) 4.53-4.77 (m, 1H)5.45-5.55 (m, 1H) 6.37 (dd, J=15.37, 6.59 Hz, 1H) 6.56 (s, 1H) 8.05 (s,1H). LCMS (ESI) 441 (M+H).

7-[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.90 (d, J=6.73 Hz, 3H) 0.96(d, J=7.03 Hz, 3H) 1.55-1.66 (m, 10H) 4.14 (dd, J=13.61, 3.95 Hz, 1H)4.52-4.63 (m, 1H) 4.84 (dd, J=13.61, 1.32 Hz, 1H) 7.37 (s, 1H) 8.95 (s,1H). LCMS (ESI) 383 (M+H).

Compound 55

Compound 55 was synthesized using an analogous synthetic sequence asthat described for Compound 44. LCMS (ESI) 265 (M+H).

Example 56 Synthesis of Compound 56

Compound 56 was synthesized using 5-bromo-2,4-dichloro-pyrimidine andCompound 17 as starting materials, and following a similar sequence ofsynthetic steps as for Compound 55. The analytical data was consistentwith that described for its antipode (Compound 55). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.88 (d, J=6.44 Hz, 6H) 1.73-1.86 (m, 1H) 3.67-3.76 (m,2H) 4.11-4.21 (m, 1H) 7.13-7.19 (m, 1H) 8.56 (s, 1H) 9.05 (s, 1H). LCMS(ESI) 265 (M+H).

Example 57 Synthesis of Compound 57

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamate

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and tert-butylN-(2-amino-2-methyl-propyl)carbamate using analogous reaction conditionsas described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.LCMS (ESI) 379 (M+H).

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.11-1.22 (m, 6H) 1.31-1.45 (m, 15H)3.10-3.24 (m, 2H) 3.51-3.76 (m, 4H) 5.60 (s, 1H) 6.94 (s, 1H) 7.33 (t,J=6.44 Hz, 1H) 8.18 (s, 1H). LCMS (ESI) 427 (M+H).

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.43 (s, 9H) 1.73 (s, 6H) 4.06 (s,2H) 7.46 (s, 1H) 9.23 (s, 1H). LCMS (ESI) 369 (M+H).

Compound 57

Compound 57 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.73 (s,6H) 3.50 (d, J=2.93 Hz, 2H) 7.25 (s, 1H) 8.46-8.55 (m, 1H) 9.07 (s, 1H).LCMS (ESI) 251 (M+H).

Example 58 Synthesis of Compound 58

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclohexyl]methyl]carbamate

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclohexyl]methyl]carbamate was synthesized using 5-bromo-2,4-dichloro-pyrimidine andIntermediate K using the analogous reaction conditions as described fortert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.18-1.54 (m, 17H) 2.23 (d,J=14.35 Hz, 2H) 3.36 (d, J=6.44 Hz, 2H) 5.82 (s, 1H) 6.93 (s, 1H) 8.22(s, 1H). LCMS (ESI) 419 (M+H).

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclohexyl]methyl]carbamate

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclohexyl]methyl]carbamate was synthesized using similar experimental conditions to thoseused in the synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.08-1.16 (m, 6H) 1.17-1.54 (m, 17H) 2.13(br. s., 2H) 3.36 (d, J=6.73 Hz, 2H) 3.50-3.69 (m, 4H) 5.72 (s, 1H) 6.94(s, 1H) 5.72 (br. s., 1H) 8.17 (s, 1H). LCMS (ESI) 467 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]cyclohexyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]cyclohexyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.37-1.54 (m, 13H) 1.75 (br. s.,4H) 2.74 (br. s., 2H) 3.78-3.84 (m, 2H) 7.44-7.51 (m, 1H) 8.23 (s, 1H)9.11 (s, 1H). LCMS (ESI) 409 (M+H).

Compound 58

Compound 58 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.28 (br.s., 2H) 1.42 (br. s., 2H) 1.70 (br. s., 4H) 1.85-1.95 (m, 2H) 2.69 (m,2H) 7.16-7.25 (m, 1H) 8.41 (br. s., 1H) 9.04 (s, 1H). LCMS 291 (M+H).

Example 59 Synthesis of Compound 59

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate

tert-butyl N-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate was synthesized using 5-bromo-2,4-dichloro-pyrimidineand Intermediate L using analogous reaction conditions as described fortert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.34 (s, 9H) 1.50-1.58 (m, 2H) 1.63-1.78(m, 4H) 1.96-2.06 (m, 2H) 3.25 (d, J=6.15 Hz, 2H) 6.71 (s, 1H) 7.18 (t,J=6.29 Hz, 1H) 8.20 (s, 1H). LCMS (ESI) 405 (M+H).

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamate

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 453 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.47 (s, 9H) 1.74 (br. s., 2H) 1.88(br. s., 2H) 2.04 (br. s., 2H) 2.41-2.45 (m, 2H) 4.06 (s, 2H) 7.45 (s,1H) 9.11 (s, 1H). LCMS (ESI) 395 (M+H).

Compound 59

Compound 59 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.72 (br.s., 2H) 1.86-1.93 (m, 2H) 1.99 (d, J=3.81 Hz, 2H) 2.40 (br. s., 2H) 3.48(d, J=2.34 Hz, 2H) 7.22 (s, 1H) 8.53 (br. s., 1H) 9.05 (s, 1H). LCMS(ESI) 277 (M+H).

Example 60 Synthesis of Compound 60

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl] carbamate

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and Intermediate Busing analogous reaction conditions as described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.The analytical data is consistent with that described for theL-enantiomer.

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-4-methyl-pentyl]carbamate

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-4-methyl-pentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 1.21-1.31 (m, 12H) 1.38-1.46 (m,11H) 1.70 (m, 1H) 3.24 (m, 2H) 3.65-3.82 (m, 4H) 4.86 (br s., 1H), 5.65(s, 1H) 5.85 (br s., 1H) 6.94 (s, 1H) 8.21 (s, 1H). LCMS (ESI) 455(M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. The analytical data was consistent with that described for theL-isomer.

Compound 60

Compound 60 was synthesized using an analogous synthetic sequence asthat described for Compound 44. The analytical data was consistent withthat described for the L-isomer.

Example 61 Synthesis of Compound 61

To a solution of Compound 60 (100 mg, 0.00024 mole) in DMF (3.0 mL) wasadded sodium hydride (60% dispersion in oil), (27.6 mg, 3 eq). Afterstirring for 15 mins, methyl iodide (30, 2 eq) was added. The contentswere stirred at room temperature for 30 mins. After the addition of satNaHCO₃, ethyl acetate was added. Separation of the organic layerfollowed by drying with magnesium sulfate and concentration under vacuumafforded the product. Analytical data was similar to the Compound 49.

Example 62 Synthesis of Compound 62

tert-butylN-[(1S,2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]carbamate

tert-butylN-[(1S,2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[(1S,2S)-2-aminocyclopentyl]carbamate with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.27 (s, 9H) 1.42-1.54 (m, 2H) 1.56-1.65(m, 2H) 1.80-1.88 (m, 1H) 1.96-2.01 (m, 1H) 3.88-3.96 (m, 1H) 4.03-4.09(m, 1H) 6.91 (d, J=8.20 Hz, 1H) 7.41 (d, J=7.32 Hz, 1H) 8.18 (s, 1H).LCMS (ESI) 391 (M+H).

tert-butylN-[(1S,2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]carbamate

tert-butylN-[(1S,2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.13 (t, 6H) 1.28 (s, 9H) 1.42-1.52 (m,2H) 1.58-1.65 (m, 2H) 1.81-1.90 (m, 1H) 1.99-2.08 (m, 1H) 3.49-3.60 (m,2H) 3.63-3.71 (m, 2H) 3.84-3.93 (m, 1H) 3.96-4.04 (m, 1H) 5.53 (s, 1H)6.96 (d, J=7.90 Hz, 1H) 7.34 (d, J=7.03 Hz, 1H) 8.14 (s, 1H). LCMS (ESI)439 (M+H).

7-[(1S,2S)-2-(tert-butoxycarbonylamino)cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S,2S)-2-(tert-butoxycarbonylamino)cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.41-1.52 (m, 9H) 1.55-1.68 (m, 1H)1.88-2.00 (m, 2H) 2.05-2.15 (m, 1H) 2.26-2.35 (m, 1H) 2.71-2.89 (m, 1H)4.01-4.16 (m, 1H) 4.28-4.45 (m, 1H) 7.41 (s, 1H) 9.11 (s, 1H). LCMS(ESI) 381 (M+H).

Compound 62

Compound 62 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.48-1.60(m, 1H) 1.88-1.98 (m, 3H) 1.99-2.08 (m, 1H) 2.66-2.75 (m, 1H) 3.63-3.74(m, 1H) 3.99-4.12 (m, 1H) 7.21 (s, 1H) 8.89 (s, 1H) 9.04 (s, 1H). LCMS(ESI) 263 (M+H).

Example 63 Synthesis of Compound 63

To chloro tricycliclactam (0.050 g, 0.225 mmole) in dioxane (2.0 mL)under nitrogen was added 5-(4-methylpiperazin-1-yl)pyridin-2-amine(0.052 g, 1.2 eq, 0.270 mmole) followed by the addition of Pd₂(dba)₃(18.5 mg), BINAP (25 mg) and sodium-tert-butoxide (31 mg, 0.324 mmole).The contents of the flask are degassed for 10 minutes and then heated to100 degrees for 12 hours. The crude reaction was loaded on a silica gelcolumn and eluted with DCM/MeOH (0-15%) to afford the desired product(26 mg). To this compound dissolved in DCM/MeOH (10%) was added 3N HClin iso-propanol (2 eq) and the reaction was stirred overnight.Concentration under vacuum afforded the hydrochloride salt. ¹HNMR(d6-DMSO) δ ppm 11.13 (brs, 1H), 9.07 (s, 1H), 8.42 (s, 1H), 8.03 (br m1H), 7.99 (s, 1H), 7.67 (brm, 1H), 7.18 (s, 1H), 4.33 (m, 2H), 3.79 (m,2H), 3.64 (m, 2H), 3.50 (m, 2H), 3.16 (m, 4H), 2.79 (s, 3H). LCMS (ESI)379 (M+H).

Example 64 Synthesis of Compound 64

To chloro tricycliclactam (0.075 g, 0.338 mmole) in dioxane (3.5 mL)under nitrogen was added tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate (0.098 g, 1.05 eq)followed by the addition of Pd₂(dba)₃ (27 mg), BINAP (36 mg) andsodium-tert-butoxide (45 mg). The contents were heated at reflux for 11hrs. The crude reaction was loaded onto a silica gel column and elutedwith DCM/MeOH (0-10%) to afford the desired product (32 mg). ¹HNMR(d6-DMSO) δ ppm 9.48 (s, 1H), 8.84 (s, 1H), 8.29 (s, 1H), 8.18 (s, 1H),7.99 (s, 1H), 7.42 (m, 1H), 6.98 (s, 1H), 4.23 (m, 2H), 3.59 (m, 2H),3.45 (m, 4H), 3.50 (m, 2H), 3.05 (m, 4H). LCMS (ESI) 465 (M+H).

Example 65 Synthesis of Compound 65

To a solution of Compound 64 (23 mg) in 10% DCM/MeOH was added 10 mL ofa 3M solution of HCl in iso-propanol. The contents were stirred for 16hrs. Concentration of the reaction mixture afforded the hydrochloridesalt. ¹HNMR (d6-DMSO) δ ppm 9.01 (s, 1H), 7.94 (m, 1H), 7.86 (m, 1H),7.23 (s, 1H), 4.30 (m, 2H), 3.64 (m, 2H), 3.36 (m, 4H), 3.25 (m, 4H).LCMS (ESI) 465 (M+H).

Example 66 Synthesis of Compound 66

To chloro-N-methyltricyclic amide (0.080 g, 0.338 mmole) in dioxane (3.5mL) under nitrogen was added tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate 0.102 g (1.1 eq) followedby the addition of Pd₂(dba)₃ (27 mg), BINAP (36 mg) andsodium-tert-butoxide (45 mg). The contents were heated at reflux for 11hrs. The crude product was purified using silica gel columnchromatography with an eluent of dichloromethane/methanol (0-5%) toafford the desired product (44 mg). ¹HNMR (d6-DMSO) δ ppm 9.49 (s, 1H),8.85 (s, 1H), 8.32 (m, 1H), 8.02 (s, 1H), 7.44 (m, 1H), 7.00 (s, 1H),4.33 (m, 2H), 3.80 (m, 2H), 3.48 (m, 4H), 3.07 (m, 4H), 3.05 (s, 3H),1.42 (s, 9H). LCMS (ESI) 479 (M+H).

Example 67 Synthesis of Compound 67

To Compound 66 (32 mg) was added 3N HCL (10 mL) in isopropanol and thecontents were stirred at room temperature overnight for 16 hrs.Concentration afforded the hydrochloride salt. ¹HNMR (d6-DMSO) δ ppm9.13 (m, 2H), 8.11 (m, 1H), 8.10 (s, 1H), 7.62 (m, 1H), 7.21 (s, 1H),4.43 (m, 2H), 3.85 (m, 2H), 3.41 (m, 4H), 3.28 (m, 4H), 3.08 (s, 3H).LCMS (ESI) 379 (M+H).

Example 68 Synthesis of Compound 68

Compound 68 was synthesized using similar experimental conditions tothat described for compound 64. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.79 (d,J=7.03 Hz, 3H) 1.01 (d, J=6.73 Hz, 3H) 1.35-1.48 (m, 9H) 2.16 (dd,J=14.64, 6.73 Hz, 1H) 3.00-3.14 (m, 4H) 3.40-3.51 (m, 4H) 3.51-3.60 (m,1H) 3.63-3.74 (m, 1H) 4.44 (dd, J=7.90, 3.81 Hz, 1H) 6.99 (s, 1H) 7.46(dd, J=8.93, 2.78 Hz, 1H) 7.94-8.09 (m, 2H) 8.31 (dd, J=9.08, 1.46 Hz,1H) 8.85 (s, 1H) 9.46 (s, 1H). LCMS (ESI) 507 (M+H).

Example 69 Synthesis of Compound 69

Compound 69 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.77-0.86 (m, 3H) 0.96 (d, J=7.03 Hz, 3H)2.10-2.24 (m, 1H) 3.07 (s, 3H) 3.37-3.79 (m, 8H) 4.00 (dd, J=13.61, 4.54Hz, 2H) 4.63-4.73 (m, 1H) 7.20 (s, 1H) 7.58-7.71 (m, 1H) 7.99 (d, J=2.34Hz, 1H) 8.12 (d, J=9.37 Hz, 1H) 9.11 (s, 1H) 9.41 (br. s., 2H) 11.76(br. s., 1H). LCMS (ESI) 421 (M+H).

Example 70 Synthesis of Compound 70

Compound 70 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. The characterization data (NMR and LCMS) was consistent with thatreported for compound 71.

Example 71 Synthesis of Compound 71

Compound 71 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.79 (d, J=6.73 Hz, 3H) 1.01 (d,J=6.73 Hz, 3H) 2.18 (dd, J=14.49, 7.17 Hz, 1H) 3.18-3.84 (m, 10H)4.53-4.71 (m, 1H) 7.24 (s, 1H) 7.65 (d, J=9.37 Hz, 1H) 8.01 (d, J=2.64Hz, 1 H) 8.14 (d, J=1.46 Hz, 1H) 8.35 (d, J=5.27 Hz, 1H) 9.14 (s, 1H)9.46 (s, 2H) 11.80 (s, 1H) LCMS (ESI) 407 (M+H).

Example 72 Synthesis of Compound 72 (Compound UUU)

Compound 72 was synthesized using similar experimental conditions tothat described for compounds 64 and 65 and was recovered as an HCl salt.¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.77 (d, J=7.03 Hz, 3H) 0.99 (d, J=6.73Hz, 3H) 2.10-2.24 (m, 1H) 3.18-3.81 (m, 10H) 4.54-4.69 (m, 1H) 7.22 (s,1H) 7.63 (d, J=9.08 Hz, 1H) 7.99 (d, J=2.63 Hz, 1H) 8.11 (s, 1H) 8.33(d, J=5.27 Hz, 1H) 9.12 (s, 1H) 9.43 (s, 2H) 11.77 (s, 1H). LCMS (ESI)407 (M+H).

Example 73 Synthesis of Compound 73

Compound 73 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.84 (d, J=6.73 Hz, 3H) 0.98 (d,J=6.73 Hz, 3H) 2.12-2.26 (m, 1H) 3.09 (s, 3H) 3.22-3.81 (m, 8H) 4.01(dd, J=13.61, 4.25 Hz, 2H) 4.59-4.72 (m, 1H) 7.19 (s, 1H) 7.74 (s, 1H)7.96-8.10 (m, 2H) 9.08 (s, 1H) 9.22 (s, 2H). LCMS (ESI) 421 (M+H).

Example 74 Synthesis of Compound 74

Compound 74 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=4.98 Hz, 3H) 0.95 (d, J=4.98 Hz, 3H)1.42-1.70 (m, 3H) 2.77 (d, J=2.93 Hz, 3H) 3.07-4.14 (m, 10H) 4.95 (s,1H) 7.20 (s, 1H) 7.66 (d, J=9.66 Hz, 1H) 7.94 (s, 1H) 8.08-8.16 (m, 1H)8.33 (d, J=4.68 Hz, 1H) 9.09 (s, 1H) 11.38 (s, 1H) 11.71 (s, 1H). LCMS(ESI) 435 (M+H).

Example 75 Synthesis of Compound 75

Compound 75 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.87 (d, J=6.15 Hz, 3H) 0.94 (d,J=6.15 Hz, 3H) 1.57 (d, J=84.61 Hz, 3H) 3.05 (s, 3H) 3.13-3.55 (m, 8H)3.69 (d, J=78.17 Hz, 2H) 4.90 (s, 1H) 7.15 (s, 1H) 7.63-7.85 (m, 1H)7.93 (s, 1H) 8.26 (s, 1H) 9.03 (s, 1H) 9.20 (s, 2H). LCMS (ESI) 421(M+H).

Example 76 Synthesis of Compound 76

Compound 76 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=6.44 Hz, 3H) 0.95 (d, J=6.44 Hz, 3H)1.43-1.70 (m, 3H) 2.78 (d, J=2.93 Hz, 3H) 3.05 (s, 3H) 3.24-3.84 (m, 8H)4.01 (d, J=9.66 Hz, 2H) 4.89-5.01 (m, 1H) 7.15 (s, 1H) 7.77 (s, 1H)7.91-8.05 (m, 2H) 9.03 (s, 1H) 10.96-11.55 (m, 2H). LCMS (ESI) 449(M+H).

Example 77 Synthesis of Compound 77

Compound 77 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.83-0.88 (d, J=6.15 Hz, 3H) 0.95(d, J=6.15 Hz, 3H) 1.40-1.71 (m, 3H) 3.28-3.83 (m, 8H) 4.00 (d, J=3.22Hz, 2H) 4.91-5.08 (m, 1H) 7.17 (s, 1H) 7.68 (d, J=9.66 Hz, 1H) 7.93 (s,1H) 8.07 (s, 1H) 9.06 (s, 1H) 9.40 (s, 2H) 11.59 (s, 1H). LCMS (ESI) 435(M+H).

Example 78 Synthesis of Compound 78

To Compound 50 0.060 g (0.205 mmole) was added5-(4-methylpiperazin-1-yl)pyridin-2-amine (35.42 mg, 0.9 eq) followed bythe addition of 1,4-dioxane (3 mL). After degassing with nitrogen,Pd₂dba₃ (12 mg), BINAP (16 mg) and sodium tert-butoxide (24 mg) wereadded. The contents were then heated at 90 degrees in a CEM Discoverymicrowave for 3 hrs. The reaction was then loaded onto a silica gelcolumn and purified by eluting with DCM/MeOH (0-15%). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.75 (t, J=7.47 Hz, 3H) 0.91 (d, J=6.73 Hz, 3H) 1.04-1.20(m, 2H) 1.80-1.98 (m, 1H) 2.77 (d, J=3.81 Hz, 3H) 2.94-3.90 (m, 10H)4.54-4.68 (m, 1H) 7.06-7.23 (m, 2H) 7.56-7.75 (m, 1H) 7.90-8.12 (m, 2H)8.29 (s, 1H) 9.07 (s, 1H) 10.98-11.74 (m, 2H). LCMS (ESI) 435 (M+H).

Example 79 Synthesis of Compound 79

Compound 79 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.75(t, J=7.32 Hz, 3H) 0.90 (d, J=6.73 Hz, 3H) 1.07-1.15 (m, 2H) 1.85-1.94(m, 1H) 3.17-3.75 (m, 10H) 4.58-4.67 (m, 1H) 7.17 (s, 1H) 7.71 (s, 1H)7.96 (s, 1H) 7.98-8.05 (m, 1H) 8.28 (d, J=4.10 Hz, 1H) 9.06 (s, 1H) 9.39(s, 2H). LCMS (ESI) 421 (M+H).

Example 80 Synthesis of Compound 80

Compound 80 was synthesized in a similar manner to that described forcompound 78. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.78 (t, J=7.32 Hz, 3H) 0.86(d, J=6.73 Hz, 3H) 1.13-1.21 (m, 2H) 1.84-1.96 (m, 1H) 2.77 (d, J=4.39Hz, 3H) 3.04 (s, 3H) 3.11-3.84 (m, 8H) 3.98 (dd, J=13.61, 4.25 Hz, 2H)4.66-4.74 (m, 1H) 7.17 (s, 1H) 7.64 (s, 1H) 7.96 (d, J=2.34 Hz, 1H)8.03-8.13 (m, 1H) 9.08 (s, 1H) 11.26 (s, 1H) 11.66 (s, 1H). LCMS (ESI)449 (M+H).

Example 81 Synthesis of Compound 81

The compound was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.78(t, J=7.32 Hz, 3H) 0.85 (d, J=6.73 Hz, 3H) 1.10-1.27 (m, 2H) 1.82-1.99(m, 1H) 3.04 (s, 3H) 3.28-3.77 (m, 8H) 3.97 (dd, J=13.91, 4.54 Hz, 2H)4.62-4.75 (m, 1H) 7.07-7.24 (m, 1H) 7.62-7.75 (m, 1H) 7.94 (d, J=2.34Hz, 1H) 7.97-8.08 (m, 1H) 9.05 (s, 1H) 9.29 (s, 2H). LCMS (ESI) 435(M+H).

Example 82 Synthesis of Compound 82

The compound was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.96(s, 9H) 3.15-3.87 (m, 10H) 4.42-4.53 (m, 1H) 6.99 (s, 1H) 7.24 (s, 1H)8.06 (s, 1H) 8.11-8.21 (m, 1H) 8.79-8.98 (m, 2H) 9.25 (s, 2H) 9.88 (s,1H). LCMS (ESI) 421 (M+H).

Example 83 Synthesis of Compound 83

Compound 83 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.95(s, 9H) 2.79 (d, J=4.10 Hz, 3H) 3.06-3.86 (m, 10H) 4.56-4.67 (m, 1H)7.17 (s, 1H) 7.70 (s, 1H) 7.96 (d, J=2.63 Hz, 1H) 7.99-8.08 (m, 1H) 8.26(s, 1H) 9.06 (s, 1H) 10.80 (s, 1H). LCMS (ESI) 435 (M+H).

Example 84 Synthesis of Compound 84

Compound 84 was synthesized in a similar manner to that described forcompound 78 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δppm 2.75-2.81 (m, 3H) 3.12-3.16 (m, 2H) 3.46-3.54 (m, 4H) 3.60-3.69 (m,2H) 3.72-3.79 (m, 1H) 4.07-4.18 (m, 2H) 6.06-6.09 (m, 1H) 6.90 (d,J=7.61 Hz, 2H) 7.20-7.31 (m, 3H) 7.33 (s, 1H) 7.49-7.55 (m, 1H)7.62-7.70 (m, 1H) 7.92 (d, J=2.93 Hz, 1H) 8.22 (s, 1H) 9.14 (s, 1H).LCMS (ESI) 455 (M+H).

Example 85 Synthesis of Compound 85

Compound 85 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 3.21(s, 4H) 3.35-3.67 (m, 5H) 4.07-4.20 (m, 2H) 6.13 (s, 1H) 6.90 (d, J=7.32Hz, 2H) 7.22-7.31 (m, 3H) 7.36 (s, 1H) 7.48 (d, J=9.37 Hz, 1H) 7.93 (d,J=2.34 Hz, 1H) 8.04-8.11 (m, 1H) 8.25 (d, J=4.98 Hz, 1H) 9.17 (s, 1H)11.77 (br, s., 1H). LCMS (ESI) 441 (M+H).

Example 86 Synthesis of Compound 86

Compound 86 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.90(d, J=6.15 Hz, 6H) 1.72-1.89 (m, 1H) 3.15-3.92 (m, 9H) 4.10-4.46 (m, 2H)7.18 (s, 1H) 7.59 (d, J=8.78 Hz, 1H) 8.00 (s, 1H) 8.13 (d, J=9.37 Hz,1H) 8.55 (s, 1H) 9.09 (s, 1H) 9.67 (s, 2H) 11.91 (s, 1H). LCMS (ESI) 407(ESI).

Example 87 Synthesis of Compound 87

Compound 87 was synthesized in a manner similar to compound 86 and wasconverted to an HCl salt. The characterization data (NMR and LCMS) wassimilar to that obtained for the antipode compound 86.

Example 88 Synthesis of Compound 88

Compound 88 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.78(s, 6H) 3.40-3.53 (m, 6H) 3.64-3.73 (m, 4H) 7.27 (s, 1H) 7.66 (d, J=9.37Hz, 1H) 7.98 (d, J=2.34 Hz, 1H) 8.12 (br. s., 1H) 8.47 (br. s., 1H) 9.11(s, 1H) 9.45 (br. s., 2H) 11.62 (br. s., 1H). LCMS (ESI) 393 (M+H).

Example 89 Synthesis of Compound 89 (also referred to as Compound T)

Compound 89 was synthesized in a similar manner to that described forcompound 78 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.47 (br. s., 6H) 1.72 (br. s., 2H) 1.92 (br. s., 2H) 2.77 (br. s.,3H) 3.18 (br. s., 2H) 3.46 (br. s., 2H) 3.63 (br. s., 2H) 3.66 (d,J=6.15 Hz, 2H) 3.80 (br. s., 2H) 7.25 (s, 1H) 7.63 (br. s., 2H) 7.94(br. s., 1H) 8.10 (br. s., 1H) 8.39 (br. s., 1H) 9.08 (br. s., 1H) 11.59(br. s., 1H). LCMS (ESI) 447 (M+H).

Example 90 Synthesis of Compound 90 (also referred to as Compound Q)

Compound 90 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.27-1.64 (m, 6H) 1.71 (br. s., 2H) 1.91 (br. s., 2H) 2.80 (br. s., 1H)3.17-3.24 (m, 2H) 3.41 (br. s., 4H) 3.65 (br. s., 4H) 7.26 (br. s., 1H)7.63 (br. s., 1H) 7.94 (br. s., 1H) 8.13 (br. s., 1H) 8.40 (br. s., 1H)9.09 (br. s., 1H) 9.62 (br. s., 1H) 11.71 (br. s., 1H). LCMS (ESI) 433(M+H).

Example 91 Synthesis of Compound 91 (also referred to as Compound ZZ)

Compound 91 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.64-1.75 (m, 2H) 1.83-1.92 (m, 2H) 1.96-2.06 (m, 2H)2.49-2.58 (m, 2H) 2.79 (d, J=3.81 Hz, 3H) 3.06-3.18 (m, 4H) 3.59-3.69(m, 2H) 3.73-3.83 (m, 2H) 4.04-4.12 (m, 2H) 7.17 (br. s., 1H) 7.60-7.70(m, 2H) 7.70-7.92 (m, 2H) 7.96 (br. s., 1H) 8.41 (br. s., 1H) 8.98 (br.s., 1H) 10.77 (br. s., 1H). LCMS (ESI) 433 (M+H).

Example 92 Synthesis of Compound 92

Compound 92 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.64-1.75 (m, 2H) 1.84-1.92 (m, 2H) 1.96-2.05 (m, 2H) 2.48-2.56 (m, 2H)3.22 (br. s., 4H) 3.42-3.48 (m, 4H) 3.60-3.69 (m, 2H) 4.05-4.13 (m, 1H)7.18 (s, 1H) 7.65 (d, J=13.47 Hz, 1H) 7.70-7.77 (m, 1H) 7.94 (d, J=1.76Hz, 1H) 8.42 (br. s., 1H) 9.00 (s, 1H) 9.15 (br. s., 2H). LCMS (ESI) 419(M+H).

Example 93 Synthesis of Compound 93

Compound 93 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.76(br. s., 2H) 1.89 (br. s., 2H) 2.03 (br. s., 2H) 2.47-2.58 (m, 2H) 3.04(s, 3H) 3.22 (br. s., 4H) 3.39 (br. s., 4H) 3.66 (s, 2H) 7.21 (s, 1H)7.67 (d, J=9.37 Hz, 1H) 7.93 (br. s., 1H) 7.98-8.09 (m, 1H) 9.04 (s, 1H)9.34 (br. s., 2H) 11.31 (br. s., 1H). LCMS (ESI) 433 (M+H).

Example 94 Synthesis of Compound 94

Compound 94 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.66-1.77 (m, 2H) 1.84-1.94 (m, 2H) 1.96-2.08 (m, 2H)2.48-2.57 (m, 2H) 3.36-3.52 (m, 4H) 3.60-3.80 (m, 6H) 7.21 (s, 1H)7.53-7.74 (m, 2H) 7.86 (s, 1H) 8.02 (s, 1H) 8.45 (s, 1H) 9.03 (s, 1H)11.19 (br. s., 1H). LCMS (ESI) 420 (M+H).

Example 95 Synthesis of Compound 95

Compound 95 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.65-1.79 (m, 2H) 1.85-1.95 (m, 2H) 1.97-2.08 (m, 2H)2.47-2.54 (m, 2H) 3.40-3.58 (m, 5H) 3.65 (dd, J=21.67, 5.56 Hz, 1H)3.69-3.78 (m, 4H) 7.24 (s, 1H) 7.97-8.17 (m, 2H) 8.48 (s, 1H) 9.08 (s,1H) 11.81 (s, 1H). LCMS (ESI) 421 (M+H).

Example 96 Synthesis of Compound 96

Compound 96 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.55-1.74 (m, 2H) 1.80-1.98 (m, 4H) 2.48-2.60 (m, 2H)3.40-3.50 (m, 4H) 3.57-3.72 (m, 2H) 3.90-4.20 (m, 4H) 7.08 (s, 1H)7.37-7.57 (m, 2H) 7.70 (m, 2H) 8.32 (s, 1H) 8.88 (s, 1H) 9.98 (s, 1H).LCMS (ESI) 419 (M+H).

Example 97 Synthesis of Compound 97 (also referred to as Compound III)

Compound 97 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.30 (d, J=5.27 Hz, 6H) 1.65-1.78 (m, 2H) 1.83-1.95 (m,2H) 1.97-2.10 (m, 2H) 2.45-2.55 (m, 2H) 3.25-3.36 (m, 1H) 3.39-3.48 (m,4H) 3.60-3.70 (m, 4H) 3.75-4.15 (m, 2H) 7.24 (s, 1H) 7.54-7.75 (m, 2H)7.95 (s, 1H) 8.10 (s, 1H) 8.49 (s, 1H) 9.07 (s, 1H) 11.25 (s, 1H) 11.48(s, 1H). LCMS (ESI) 461 (M+H).

Example 98 Synthesis of Compound 98

Compound 98 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.99 (d, J=6.15 Hz, 6H) 1.65-1.78 (m, 2H) 1.90 (m, 2H)1.97-2.08 (m, 2H) 2.08-2.17 (m, 1H) 2.45-2.55 (m, 2H) 2.88-3.02 (m, 2H)3.33-3.48 (m, 4H) 3.50-3.90 (m, 6H) 7.24 (s, 1H) 7.67 (s, 2H) 7.94 (s,1H) 8.12 (s, 1H) 8.49 (s, 1H) 9.07 (s, 1H) 10.77 (s, 1H) 11.51 (s, 1H).LCMS (ESI) 475 (M+H).

Example 99 Synthesis of Compound 99

Compound 99 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.13 (d, J=5.86 Hz, 6H) 1.66-1.77 (m, 2H) 1.84-1.94 (m,2H) 1.97-2.09 (m, 2H) 2.40-2.53 (m, 2H) 3.37-3.49 (m, 2H) 3.50-3.59 (m,2H) 3.59-3.73 (m, 4H) 7.23 (s, 1H) 7.64 (m, 3H) 7.85 (s, 1H) 8.11 (s,1H) 8.47 (s, 1H) 9.05 (s, 1H). 11.35 (br s., 1H). LCMS (ESI) 448 (M+H).

Example 100 Synthesis of Compound 100

Compound 100 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.50-1.57 (m, 2H) 1.62-1.68 (m, 3H) 1.68-1.75 (m, 2H)1.84-1.92 (m, 2H) 1.97-2.08 (m, 2H) 2.48-2.53 (m, 2H) 3.14-3.23 (m, 4H)3.43-3.47 (m, 2H) 3.58-3.70 (m, 2H) 7.22 (s, 1H) 7.58-7.70 (m, 2H)7.85-8.00 (m, 1H) 8.16 (d, 1H) 8.46 (s, 1H) 9.04 (s, 1H) 11.37 (br s.,1H). LCMS (ESI) 418 (M+H).

Example 101 Synthesis of Compound 101 (also referred to as Compound WW)

Compound 101 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.72 (s, 2H) 1.90 (s, 4H) 2.03 (s, 2H) 2.21 (s, 2H)2.48-2.54 (m, 2H) 2.73 (s, 2H) 3.03 (s, 2H) 3.25-3.35 (m, 1H) 3.38-3.48(m, 4H) 3.65-3.99 (m, 5H) 7.23 (s, 1H) 7.63 (d, J=9.66 Hz, 1H) 7.90 (s,1H) 8.13 (s, 1H) 8.47 (s, 1H) 9.06 (s, 1H) 10.50 (br s., 1H). LCMS (ESI)503 (M+H).

Example 102 Synthesis of Compound 102 (Also Referred to as Compound HHH)

Compound 102 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.63-1.85 (m, 6H) 1.87-1.92 (m, 2H) 1.99-2.06 (m, 2H)2.15-2.23 (m, 2H) 2.47-2.53 (m, 1H) 2.69-2.79 (m, 2H) 2.81-2.91 (m, 2H)2.98-3.08 (m, 2H) 3.32-3.48 (m, 4H) 3.57-3.72 (m, 4H) 3.77-3.85 (m, 2H)7.22 (s, 1H) 7.60-7.68 (m, 2H) 7.90 (s, 1H) 8.07 (s, 1H) 8.46 (s, 1H)9.04 (s, 1H). 11.41 (br s., 1H). LCMS (ESI) 501 (M+H).

Example 103 Synthesis of Compound 103

Compound 103 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.64-1.76 (m, 2H) 1.87-1.93 (m, 2H) 2.00-2.07 (m, 2H)2.48-2.53 (m, 2H) 2.67-2.72 (m, 4H) 3.44-3.47 (m, 2H) 3.50-3.55 (m, 4H)7.24 (s, 1H) 7.61 (d, J=9.37 Hz, 2H) 7.86 (d, J=2.63 Hz, 1H) 8.09 (d,J=12.88 Hz, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 11.41 (br s., 1H). LCMS (ESI)436 (M+H).

Example 104 Synthesis of Compound 104

Compound 104 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.29 (d, J=6.73 Hz, 6H) 1.66-1.79 (m, 2H) 1.84-1.95 (m,2H) 1.98-2.09 (m, 2H) 2.46-2.55 (m, 2H) 3.29-3.39 (m, 2H) 3.58-3.70 (m,4H) 3.77-3.86 (m, 4H) 7.24 (s, 1H) 7.66 (d, J=9.37 Hz, 1H) 7.96 (d,J=2.93 Hz, 1H) 8.08 (s, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 9.28 (s, 1H) 9.67(s, 1H) 11.36 (s, 1H). LCMS (ESI) 447 (M+H).

Example 105 Synthesis of Compound 105

Compound 105 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.73 (s, 2H) 1.76-1.85 (m, 2H) 1.85-1.94 (m, 2H)1.98-2.07 (m, 2H) 2.19-2.26 (m, 2H) 2.48-2.52 (m, 1H) 2.70-2.81 (m, 4H)3.13-3.20 (m, 1H) 3.30-3.48 (m, 3H) 3.58-3.71 (m, 4H) 3.78-3.84 (m, 4H)7.24 (s, 1H) 7.62 (d, J=9.37 Hz, 2H) 7.89 (d, J=1.17 Hz, 1H) 8.09-8.18(m, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 11.46 (br s., 1H). LCMS (ESI) 519(M+H).

Example 106 Synthesis of Compound 106

Compound 106 was synthesized using similar conditions to those describedfor compound 78 followed by the deblocking step described for compound65 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.65-1.75 (m, 2H) 1.85-1.93 (m, 2H) 1.93-1.99 (m, 1H) 2.00-2.06 (m, 2H)2.08-2.14 (m, 1H) 2.47-2.55 (m, 2H) 3.07-3.25 (m, 2H) 3.25-3.69 (m, 5H)4.46 (s, 1H) 4.67 (s, 1H) 7.22 (s, 1H) 7.58-7.69 (m, 2H) 8.46 (s, 1H)9.02 (s, 1H) 9.34 (s, 1H) 9.65 (s, 1H). LCMS (ESI) 431 (M+H).

Example 107 Synthesis of Compound 107 (also referred to as Compound YY)

Compound 107 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.65-1.82 (m, 3H) 1.89 (br. s., 2H) 1.98-2.08 (m, 2H)2.13 (br. s., 2H) 2.47-2.55 (m, 2H) 2.68 (d, J=4.98 Hz, 6H) 2.71-2.80(m, 2H) 3.29-3.71 (m, 10H) 7.16-7.26 (m, 1H) 7.67 (d, J=9.66 Hz, 2H)7.91 (d, J=2.05 Hz, 1H) 8.14 (br. s., 1H) 8.48 (br. s., 1H) 9.05 (s, 1H)11.14 (br. s., 1H) 11.43 (br. s., 1H). LCMS (ESI) 461 (M+H).

Example 108 Synthesis of Compound 108

Compound 108 was synthesized in a manner similar to that described forcompounds 64 and 65 and was recovered as an HCl salt. The analyticaldata was consistent with that described for the antipode compound 75.

Example 109 Synthesis of Compound 109

Compound 109 was synthesized in a manner similar to that described forcompounds 64 and 65 and was recovered as an HCl salt. The analyticaldata was consistent with that described for the antipode compound 75.

Example 110 Synthesis of Compound 110

Compound 110 was synthesized in a similar manner to that described forcompound 78 and then converted to its hydrochloride salt. ¹HNMR (600MHz, DMSO-d₆) δ ppm 1.50-1.65 (m, 1H) 1.92-2.02 (m, 3H) 2.06-2.15 (m,1H) 2.78 (d, J=3.81 Hz, 4H) 3.10-3.20 (m, 4H) 3.47-3.51 (m, 2H)3.64-3.71 (m, 1H) 3.76-3.83 (m, 2H) 3.98-4.14 (m, 1H) 7.20 (s, 2H) 7.77(s, 1H) 7.97 (s, 2H) 8.81 (s, 1H) 9.03 (s, 1H) 10.97 (br s., 1H). LCMS(ESI) 419 (M+H).

Example 111 Synthesis of Compound 111

Compound 111 was synthesized in a similar manner to that described forcompound 78 and then converted to its hydrochloride salt. ¹HNMR (600MHz, DMSO-d₆) δ ppm 1.54-1.59 (m, 1H) 1.92-2.01 (m, 3H) 2.06-2.15 (m,1H) 2.76-2.84 (m, 1H) 3.17-3.24 (m, 6H) 3.64-3.71 (m, 2H) 4.02-4.11 (m,2H) 7.22 (s, 2H) 7.64 (s, 1H) 7.97 (s, 2H) 8.75 (s, 1H) 8.97 (s, 1H)9.21 (s, 1H). LCMS (ESI) 405 (M+H).

Example 112 Synthesis of Compound 112

Compound 112 was synthesized using similar experimental conditions tothat described for compound 64.

Example 113 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate, Compound113

To a solution of 5-bromo-2,4-dichloropyrimidine (12.80 g, 0.054 mole) inethanol (250 mL) was added Hunig's base (12.0 mL) followed by theaddition of a solution of N-(tert-butoxycarbonyl)-1,2-diaminoethane (10g, 0.0624 mole) in ethanol (80 mL). The contents were stirred overnightfor 20 hrs. The solvent was evaporated under vacuum. Ethyl acetate (800mL) and water (300 mL) were added and the layers separated. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum. Column chromatography on silica gel using hexane/ethyl acetate(0-60%) afforded tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)351 (M+H).

Example 114 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate, Compound 114

To tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate (5 g, 14.23mmole) in toluene (42 mL) and triethylamine (8.33 mL) under nitrogen wasadded triphenyl arsine (4.39 g), 3,3-diethoxyprop-1-yne (3.24 mL) andPddba (1.27 g). The contents were heated at 70 degrees for 24 hrs. Afterfiltration through CELITE®, the crude reaction was columned usinghexane/ethyl acetate (0-20%) to afford the desired product 3.9 g).Column chromatography of the resulting residue using hexane/ethylacetate (0-30%) afforded tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.LCMS (ESI) 399 (M+H).

Example 115 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 115

To a solution of Compound 114 (3.9 g, 0.00976 mole) in THE (60 mL) wasadded TBAF (68.3 mL, 7 eq). The contents were heated to 45 degrees for 2hrs. Concentration followed by column chromatography using ethylacetate/hexane (0-50%) afforded tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale brown liquid (1.1 g). ¹HNMR (d6-DMSO) δ ppm 8.88 (s, 1H), 6.95(brs, 1H), 6.69 (s, 1H), 5.79 (s, 1H), 4.29 (m, 2H), 3.59 (m, 4H), 3.34(m, 1H), 3.18 (m, 1H), 1.19 (m, 9H), 1.17 (m, 6H). LCMS (ESI) 399 (M+H).

Example 116 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 116

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.1 g, 0.00025 mol) in acetonitrile (2 mL) was added1,3-diiodo-5,5-dimethylhydantoin (95 mg, 1 eq), and solid NaHCO₃ (63 mg,3 eq). The reaction was stirred at room temperature for 16 hrs. Thereaction was filtered and concentrated in vacuo. The product waspurified by silica gel column chromatography using hexane/ethylacetate(0-50%) to afford tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale yellow solid (0.03 g). LCMS (ESI) 525 (M+H).

Example 117 Synthesis of tert-ButylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 117

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.1 g, 0.19 mmole) in dioxane (3 mL) was added 2-methylphenylboronicacid (28 mg), tetrakis(triphenylphosphine)palladium (25 mg) andpotassium phosphate (250 mg) in water (0.3 mL). The reaction was heatedin a CEM Discovery microwave at 90° C. for 3 hrs. The crude reaction wasloaded onto silica gel and columned using hexane/ethyl acetate (0-30%)to afford tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.06 g). LCMS (ESI) 489 (M+H).

Example 118 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 118

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.85 g, 1.74 mmole) in AcOH (10 mL) was added water (1.5 mL). Thereaction was stirred at room temperature for 16 hrs. The crude reactionwas then concentrated under vacuum. After the addition of ethyl acetate(50 mL), the organic layer was washed with satd. NaHCO₃. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum to afford the crude intermediate, tert-butylN-[2-[2-chloro-6-formyl-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.To this crude intermediate in DMF (5 mL) was added oxone (1.3 g). Afterstirring for 2.5 hrs, water (20 mL) and ethyl acetate (100 mL) wereadded. The organic layer was separated, dried and then concentratedunder vacuum to afford the crude product which was columned over silicagel using hexane/ethyl acetate (0-50%) to afford7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.112 g). LCMS (ESI) 431 (M+H).

Example 119 Synthesis of Compound 119

To7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.1 g, 0.261 mmol) in DCM (4.1 mL) was added DMAP (20 mg) followedby the addition of N,N′-diisopropylcarbodiimide (0.081 mL, 2 eq). Afterstirring for 3 hrs, TFA (0.723 mL) was added. Stirring was thencontinued for another 30 minutes. The reaction mixture was neutralizedwith satd. NaHCO₃. DCM (20 mL) was then added and the organic layerseparated, dried with magnesium sulfate and then concentrated undervacuum to afford the crude product which was columned usinghexane/ethylacetate (0-100%) to afford chloro tricyclic amide Compound119 (0.65 g). LCMS (ESI) 313 (M+H).

Example 120 Synthesis of Compound 120

To the chloro tricyclic amide (0.040 g, 0.128 mmole) (Compound 119) indioxane (2.5 mL) under nitrogen was added Pd₂(dba)₃ (12 mg), sodiumtert-butoxide (16 mg), BINAP (16 mg) and 4-morpholinoaniline (22.7 mg, 1eq). The reaction mixture was heated at 90° C. in a CEM Discoverymicrowave for 3.0 hrs. The crude reaction was loaded onto a silica gelcolumn and the contents eluted with DCM/MeOH (0-6%) to afford theproduct (10 mg). LCMS (ESI) 455 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δ ppm2.14 (s, 3H) 3.23-3.50 (m, 2H) 3.57-3.73 (m, 2H), 3.81-3.92 (m, 8H),7.11-7.31 (m, 4H) 7.31-7.48 (m, 1H) 7.58-7.73 (m, 1H) 7.77-7.95 (m, 2H)8.05-8.21 (m, 1H) 8.44 (s, 1H) 9.85-10.01 (m, 1H).

Example 121 Synthesis of Compound 121

To the chloro tricyclic amide (0.024 g) (Compound 119) inN-methyl-2-pyrrolidone (NMP) (1.5 mL) was addedtrans-4-aminocyclohexanol (0.0768 mmol, 26.54 mg, 3 eq) and Hunig's base(0.4 mL). The reaction was heated in a CEM Discovery microwave vessel at150° C. for 1.2 hrs. The crude reaction was loaded onto a silica gelcolumn and the contents eluted with DCM/MeOH (0-10%) to afford theproduct (21 mg). LCMS (ESI) 392 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.23 (d, J=8.78 Hz, 4H) 1.84 (br. s., 4H) 2.11 (s, 3H) 3.34-3.43 (m, 1H)3.55 (br. s., 2H) 3.72 (br. s., 1H) 4.13 (br. s., 2H) 4.50 (br. s., 1H)7.03 (br. s., 1H) 7.12-7.28 (m, 4H) 7.96 (br. s., 1H) 8.18 (br. s., 1H).

Example 122 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 122

7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 341 (M+H).

Example 123 Synthesis of Compound 123

Chloro tricyclic amide, Compound 123, was synthesized using a similarexperimental procedure as that described for the synthesis of chlorotricyclic amide (Compound 119). LCMS (ESI) 223 (M+H).

Example 124 Synthesis of Compound 124

To the chloro tricyclic amide, Compound 123 (0.035 g, 0.00157 mole) inNMP (1.5 mL) was added Hunig's base (0.3 mL) followed by the addition ofthe trans-4-aminocyclohexanol (54.2 mg). The reaction mixture was heatedat 150° C. for 1.5 hrs. The crude reaction was loaded onto a silica gelcolumn and the column was eluted with DCM/MeOH (0-10%) to afford theproduct (5 mg). LCMS (ESI) 302 (M+H).

Example 125 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamate,Compound 125

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-(2-amino-2-methyl-propyl)carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)(M+H) 379.

Example 126 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate,Compound 126

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewas synthesized by treating tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate. LCMS (ESI) (M+H) 427.

Example 127 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-2-methyl-propyl]carbamate,Compound 127

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-2-methyl-propyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.LCMS (ESI) (M+H) 427.

Example 128 Synthesis of7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 128

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 369 (M+H).

Example 129 Synthesis of Compound 129

Chloro tricyclic amide, Compound 129, was synthesized using a similarprocedure as that described for the synthesis of chloro tricyclic amide,Compound 119. LCMS (ESI) 251 (M+H).

Example 130 Synthesis of Compound 130

Compound 130 was synthesized by treating chlorotricyclic amine Compound129 with trans-4-aminocyclohexanol using similar experimental conditionsas for compound 124. LCMS (ESI) 330 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δppm 1.07-1.34 (m, 4H) 1.47-2.05 (m, 10H) 3.09 (m, 1H) 3.51 (d, J=2.91Hz, 2H) 3.57 (m, 1H) 4.50 (br. s., 1H) 6.89 (s, 1H) 6.94-7.05 (m, 1H)8.04 (br. s., 1H) 8.60 (s, 1H) 9.00 (br. s., 1H).

Example 131 Synthesis of benzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]carbamate,Compound 131

BenzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine with benzylN-[1-(aminomethyl)propyl]carbamate using similar experimental conditionsas described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)(M+H) 413.

Example 132 Synthesis of benzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamate,Compound 132

BenzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamatewas prepared by treating benzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]-carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamateLCMS (ESI) (M+H) 461.

Example 133 Synthesis of benzylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]propyl]carbamate,Compound 133

BenzylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]propyl]carbamatewas synthesized by treating benzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) (M+H) 461.

Example 134 Synthesis of7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 134

7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 403 (M+H).

Example 135 Synthesis of Compound 135

To a solution of7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid in dichloromethane was added HBr, the reaction was stirred at 45degrees for 3 hrs. After concentration, 2N NaOH was added to basify(pH=8.0) the reaction followed by the addition of THE (20 mL). Boc₂O wasthen added (1.2 eq) and the reaction was stirred for 16 hrs. To thecrude reaction mixture was then added ethyl acetate (100 mL) and water(50 mL) and the organic phase was separated, dried (magnesium sulfate)and then concentrated under vacuum. To the crude product was addeddichloromethane (30 mL) followed by DIC and DMAP. After stirring for 2hrs, TFA was added and the contents stirred for an hour. The solventswere evaporated under vacuum and the residue basified with satd. NaHCO₃.Ethyl acetate was then added and the organic layer separated, dried(magnesium sulfate) and then concentrated under vacuum. Columchromatography with hexane/ethyl acetate (0-100%) afforded the desiredchlorotricyclic core, Compound 135. LCMS (ESI) 251 (M+H).

Example 136 Synthesis of Compound 136

Compound 136 was synthesized by treating chlorotricyclic amine, Compound135, with trans-4-aminocyclohexanol using similar experimentalconditions as for compound 124. LCMS (ESI) 330 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.80-0.95 (m, 3H) 1.35-1.92 (m, 10H) 3.66 (br. m., 3H)4.17 (br. s., 2H) 4.47 (br. s., 1H) 6.85 (s, 1H) 6.96 (br. s., 1H) 8.15(br. s., 1H) 8.62 (br. s., 1H).

Example 137 Synthesis of tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamate,Compound 137

tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-[1-(aminomethyl)cyclopentyl]carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)405 (M+H).

Example 138 Synthesis of tert-butylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]cyclopentyl]carbamate,Compound 138

tert-butylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamatewith with 3,3-diethoxyprop-1-yne in the presence of a catalyst such asPddba using similar experimental conditions as described for thesynthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate LCMS (ESI) 453 (M+H).

Example 139 Synthesis of tert-butylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]cyclopentyl]carbamate,Compound 139

tert-butylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) 453 (M+H).

Example 140 Synthesis of7-[[1-(tert-butoxycarbonylamino)cyclopentyl]methyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 140

7-[[1-(tert-butoxycarbonylamino)cyclopentyl]methyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 395 (M+H).

Example 141 Synthesis of Compound 141

Chlorotricyclic core Compound 141 was synthesized using a similarexperimental procedure as that described for the synthesis of chlorotricyclic amide Compound 119. LCMS (ESI) 277 (M+H).

Example 142 Synthesis of Compound 142

Compound 142 was synthesized by treating chlorotricyclic amine, Compound141, with trans-4-aminocyclohexanol using similar experimentalconditions as for Compound 124. LCMS (ESI) 356 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.08-1.32 (m, 8H) 1.60-2.09 (m, 8H) 3.03-3.17 (m, 1H)3.35 (s, 2H) 3.54-3.62 (m, 1H) 4.51 (d, J=4.39 Hz, 1H) 6.88 (s, 1H) 6.96(br. s., 1H) 8.07 (br. s., 1H) 8.58 (s, 1H).

Example 143 Synthesis of tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate,Compound 143

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-[(1-aminocyclopentyl)methyl]carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)405 (M+H).

Example 144 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate,Compound 144

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamatewas synthesized by treating tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate.

LCMS (ESI) 453 (M+H).

Example 145 Synthesis of tert-butylN-[[1-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentyl]methyl]carbamate,Compound 145

tert-ButylN-[[1-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentyl]methyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) 4534 (M+H).

Example 146 Synthesis of7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6carboxylicacid, Compound 146

7-[2-(tert-Butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 395 (M+H).

Example 147 Synthesis of Compound 147

Chloro tricyclic amide, Compound 147 was synthesized using a similarexperimental procedure as that described for the chloro tricyclic amide,Compound 119. LCMS (ESI) 277 (M+H).

Example 148 Synthesis of Compound 148

Compound 148 was synthesized by treating chlorotricyclic amine, Compound147, with trans-4-aminocyclohexanol using similar experimentalconditions as for Compound 124. LCMS (ESI) 356 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.06-1.35 (m, 8H) 1.45-1.95 (m, 8H) 3.10 (m, 1H) 3.58(br. s., 2H) 3.95 (br. s., 1H) 4.49 (br. s., 1H) 6.84 (s, 1H) 6.85-6.93(m, 1H) 8.29 (s, 1H) 8.61 (br. s., 1H).

Example 149 Synthesis of Compound 149

Step 1: Compound 59 is Boc protected according to the method of A.Sarkar et al. (JOC, 2011, 76, 7132-7140).Step 2: Boc-protected Compound 59 is treated with 5 mol % NiCl₂(Ph₃)₂,0.1 eq triphenylphosphine, 3 eq Mn, 0.1 eq tetraethylammonium iodide, inDMI under CO₂ (1 atm) at 25° C. for 20 hours to convert the aryl halidederivative into the carboxylic acid.Step 3: The carboxylic acid from Step 2 is converted to thecorresponding acid chloride using standard conditions.Step 4: The acid chloride from Step 3 is reacted with N-methylpiperazine to generate the corresponding amide.Step 5: The amide from Step 4 is deprotected using trifluoroacetic acidin methylene chloride to generate the target compound. Compound 149 ispurified by silica gel column chromatography eluting with adichloromethane-methanol gradient to provide Compound 149.

Each of Compounds 119 through 147 and corresponding compounds withvarious R⁸, R¹ and Z definitions may be reacted with sodium hydride andan alkyl halide or other halide to insert the desired R substitutionprior to reaction with an amine, such as described above for thesynthesis of Compound 120, to produce the desired product of Formulae I,II, III, IV, or V.

Example 150 CDK4/6 Inhibition In Vitro Assay

Selected compounds disclosed herein were tested in CDK4/cyclinD1,CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara,Calif.) to determine their inhibitory effect on these CDKs. The assayswere performed using microfluidic kinase detection technology (CaliperAssay Platform). The compounds were tested in 12-point dose-responseformat in singlicate at Km for ATP. Phosphoacceptor substrate peptideconcentration used was 1 μM for all assays and Staurosporine was used asthe reference compound for all assays. Specifics of each assay are asdescribed below:

CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM;Incubation time: 3 hr.

CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM;Incubation time: 1 hr.

CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM;Incubation time: 10 hr.

The inhibitory IC₅₀ values for the compounds in Table 1 for CDK4/CycD1,CDK2/CycE, CDK2/CycA, as well as fold selectivity are presented in Table2.

TABLE 2 Selective Inhibition of CDK4 CDK4/ CDK2/ CycD1 CycE IC₅₀ FoldSelectivity CDK2/CycA Fold Selectivity Structure IC₅₀ [nM] [nM](CDK2/CycE/CDK4) IC₅₀ [nM] (CDK2/CycA/CDK4) A 4.2 6350 1516 3160 754 B0.4 3040 6862 1890 4266 C 1.4 1920 1333 616 428 D 0.9 3480 3779 15001629 E 1 695 688 204 202 F 1.5 628 419 190 127 G 1.5 2580 1767 646 442 H1.5 1520 1013 377 251 I 2 2120 1065 1130 568 J 0.7 5110 7707 4340 6546 K1 1070 1019 738 703 L 5.7 4530 789 1490 260 M 2.3 2280 1004 1410 621 N 11500 1500 ND ND O 2.5 41410 1636 3150 1245 P 3.3 3560 1085 1010 308 Q0.6 1080 1722 3030 4833 R 0.5 1920 3918 1360 2776 S 1.7 1250 718 342 197T 0.8 1660 2022 1670 2034 U 0.7 1460 2229 857 1308 V 2.9 3500 1224 2130745 W 2.7 3970 1481 539 201 X 0.9 11600 12975 1840 2058 Y 2.5 124 50 6125 Z 3.2 3710 1174 647 205 AA 0.5 6100 13319 4630 10109 BB 0.8 1680 2017502 603 CC 1.6 1250 791 755 478 DD 1.9 9620 5200 8360 4519 EE 3.8 1660432 1110 289 FF 1.2 4620 3949 1400 1197 GG 1 3580 3377 1510 1425 HH 1.71280 766 265 159 II 2 367 184 239 120 JJ 1.4 288 204 ND ND KK 2.3 1760762 915 396 LL 2 202 103 108 55 MM 1.8 3390 1863 597 328 NN 3.7 47001274 1560 423 OO 9 3980 442 570 63 PP 3.1 3600 1146 3090 984 QQ 4.1 3060746 2570 627 RR 1.2 1580 1374 693 603 SS 0.8 1460 1865 1390 1775 TT 0.81260 1550 596 733 UU 7.3 3960 542 ND ND VV 3.3 2630 809 789 243 WW 0.71350 204 ND ND XX 1.3 7300 5615 6290 4838 YY 4.6 6900 1490 ND ND ZZ 10.59960 949 ND ND AAA 2.3 6010 2591 2130 918 BBB 2.8 187 68 85 31 CCC 22170 1074 457 226 DDD 9.5 9350 986 ND ND EEE 0.2 2950 1266 943 405 FFF4.7 4540 966 1370 291 GGG 13.7 7610 555 ND ND HHH 6.8 2840 419 ND ND III6 3770 626 ND ND JJJ 3.2 5200 1620 2830 882 KKK 1.3 291 231 87.3 69 LLL3.2 1620 509 4530 1425 MMM 3.2 1890 600 990 314 NNN 1.4 2930 2154 1010743 OOO 2.4 393 164 203 85 PPP 0.8 16500 21263 2640 3402 QQQ 10.5 111001057 ND ND RRR 2.6 4500 1758 ND ND SSS 2 2280 1112 1880 917 TTT 3.4 3030899 ND ND UUU 18 16460 914 ND ND VVV 7.4 4380 589 ND ND WWW 18.5 2500135 ND ND XXX 11.4 6620 581 ND ND

To further characterize its kinase activity, Compound T was screenedagainst 456 (395 non-mutant) kinases using DiscoveRx's KINOMEscan™profiling service. The compound was screened using a singleconcentration of 1000 nM (>1000 times the IC50 on CDK4). Results fromthis screen confirmed the high potency against CDK4 and high selectivityversus CDK2. Additionally, the kinome profiling showed that Compound Twas relatively selective for CDK4 and CDK6 compared to the other kinasestested. Specifically, when using an inhibitory threshold of 65%, 90%, or99%, Compound T inhibited 92 (23.3%), 31 (7.8%) or 6 (1.5%) of 395non-mutant kinases respectively.

In addition to CDK4 kinase activity, several compounds were also testedagainst CDK6 kinase activity. The results of the CDK6/CycD3 kinaseassays, along with the CDK4/cyclinD1, CDK2/CycA and CDK2/cyclinE kinaseassays, are shown for PD0332991 (Reference) and the compounds T, Q, GG,and U in Table 3. The IC₅₀ of 10 nM for CDK4/cyclinD1 and 10 uM forCDK12/CyclinE agrees well with previously published reports forPD0332991 (Fry et al. Molecular Cancer Therapeutics (2004)3(11)1427-1437; Toogood et al. Journal of Medicinal Chemistry (2005) 48,2388-2406). Compounds T, Q, GG, and U are more potent (lower IC₅₀) withrespect to the reference compound (PD0332991) and demonstrate a higherfold selectivity with respect to the reference compound (CDK2/CycE IC₅₀divided by CDK4/CycD1 IC₅₀).

TABLE 3 Inhibition of CDK kinases by Compounds T, Q, GG, and UCDK4/CycD1 CDK2/CycE Fold Selectivity CDK2/CycA CDK6/CycD3 FormulaIC₅₀(nM) IC₅₀(uM) CDK2/CDK4 IC₅₀(uM) IC₅₀(nM) PD0332991 10 10 1000 NotNot determined Reference determined Compound T 0.821 1.66 2022 1.67 5.64Compound Q 0.627 1.08 1722 3.03 4.38 Compound GG 1.060 3.58 3377 1.514.70 Compound U 0.655 1.46 2229 .857 5.99

Example 151 G1 Arrest (Cellular G1 and S-Phase) Assay

To demonstrate the ability to synchronize the cell cycle of Rb-positivecells following administration of a CDK4/6 inhibitor, HS68 cells (humanskin fibroblast cell line (Rb-positive)) were stained with propidiumiodide staining solution and run on Dako Cyan Flow Cytometer. Thefraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phaseDNA cell cycle was determined using FlowJo 7.2 0.2 analysis.

The compounds listed in Table 1 were tested for their ability to arrestHS68 cells at the G1 phase of the cell cycle. From the results of thecellular G1 arrest assay, the range of the inhibitory EC₅₀ valuesnecessary for G1 arrest of HS68 cells was from 22 nM to 1500 nM (seecolumn titled “Cellular G1 Arrest EC₅₀” in Table 4).

Example 152 Inhibition of Cellular Proliferation

Cellular proliferation assays were further conducted using the followingcancer cell lines: MCF7 (breast adenocarcinoma—Rb-positive), ZR-75-1(breast ductal carcinoma—Rb-positive), H69 (human small cell lungcancer—Rb-negative) cells, or A2058 (human metastatic melanomacells—Rb-negative). These cells were seeded in Costar (Tewksbury, Mass.)3093 96 well tissue culture treated white walled/clear bottom plates.Cells were treated with the compounds of Table 1 as nine point doseresponse dilution series from 10 uM to 1 nM. Cells were exposed tocompounds and then cell viability was determined after either four (H69)or six (MCF7, ZR75-1, A2058) days as indicated using the CellTiter-Glo®luminescent cell viability assay (CTG; Promega, Madison, Wis., UnitedStates of America) following the manufacturer's recommendations. Plateswere read on BioTek (Winooski, Vt.) Syngergy2 multi-mode plate reader.The Relative Light Units (RLU) were plotted as a result of variablemolar concentration and data was analyzed using Graphpad (LaJolla,Californaia) Prism 5 statistical software to determine the EC₅₀ for eachcompound.

The results of the cellular inhibition assays for the two Rb-positivebreast cancer cell lines (MCF7 and ZR75-1) are shown in Table 4. Therange of the inhibitory EC₅₀ values necessary for inhibition of MCF7breast cancer cell proliferation was 28 nM to 257 nM. The range of theinhibitory EC₅₀ values necessary for inhibition ofZR75-1 breast cancercell proliferation was 24 nM to 581 nM.

In addition to breast cancer cell lines, a number of the compoundsdisclosed herein were also evaluated against a small cell lung cancercell line (C69) and a human metastatic melanoma cell line (A2058), twoRb-deficient (Rb-negative) cell lines. The results of these cellularinhibition assays are shown in Table 4. The range of the inhibitory EC₅values necessary for inhibition of H69 small cell lung cancer cells was2040 nM to >3000 nM. The range of the inhibitory EC₅₀ values necessaryfor inhibition of A2058 malignant melanoma cell proliferation was 1313nM to >3000 nM. In contrast to the significant inhibition seen on thetwo Rb-positive breast cancer cell lines, it was found that thecompounds tested were not significantly effective at inhibitingproliferation of the small cell lung cancer or melanoma cells.

TABLE 4 Inhibition of Cancer Cell Proliferation Cellular MCF7 ZR75-1 H69A2058 G1 Arrest Cellular Cellular Cellular Cellular EC₅₀ EC₅₀ EC₅₀ EC₅₀EC₅₀ Structure (nM) [nM] [nM] [nM] [nM] A 110 75 44 >3000 ND B 90 201245 ND ND C 95 88 73 ND ND D 50 57 46 2911 1670 E 75 53 62 2580 1371 F175 ND ND ND ND G 175 ND ND ND ND H 85 85 120 2040 1313 I 80 61 40 29501062 J 110 70 82 >3000 >3000 K 28 43 ND >3000 1787 L 65 506 ND2161 >3000 M 100 ND ND ND ND N 25 28 24 >3000 1444 O 40 56 29 >3000 2668P 30 60 43 >3000 >3000 Q 100 49 35 >3000 2610 R 70 36 50 >3000 2632 S150 76 ND >3000 >3000 T 100 49 36 >3000 >3000 U 25 70 59 >3000 >3000 V70 50 29 >3000 1353 W 160 294 ND >3000 >3000 X 65 ND ND >3000 >3000 Y350 ND ND ND ND Z 110 141 54 ND ND AA 70 47 47 >3000 ND BB 75 ND ND 29431635 CC 90 50 38 >3000 >3000 DD 100 ND ND ND ND EE 125 216 203 ND ND FF80 140 ND ND ND GG 80 52 62 2920 2691 HH 110 ND ND ND ND II 40 9433 >3000 >3000 JJ 90 122 ND >3000 >3000 KK 22 333 ND 2421 1379 LL 125 96ND >3000 >3000 MM 100 73 77 >3000 >3000 NN 110 ND ND ND ND OO 95 120229 >3000 >3000 PP 100 164 66 ND ND QQ 120 ND ND >3000 >3000 RR 90 72 ND2888 1617 SS 80 94 53 2948 1658 TT 75 ND ND ND ND UU 300 ND ND ND ND VV200 ND ND ND ND WW 400 ND ND ND ND XX 225 ND ND ND ND YY 175 257 581 NDND ZZ 500 ND ND ND ND AAA 275 320 ND >3000 >3000 BBB 230 123ND >3000 >3000 CCC 250 ND ND ND ND DDD 350 ND ND ND ND EEE 250 453ND >3000 >3000 FFF 650 ND ND ND ND GGG 350 ND ND ND ND HHH 250 ND ND NDND III 250 ND ND ND ND JJJ 240 ND ND ND ND KKK 190 ND ND ND ND LLL 250ND ND ND ND MMM 200 134 141 >3000 >3000 NNN 210 ND ND ND ND OOO 200 138ND >3000 >3000 PPP 275 ND ND ND ND QQQ 500 ND ND ND ND RRR 400 ND ND NDND SSS 1500 ND ND ND ND TTT 350 ND ND ND ND UUU 300 ND ND ND ND VVV 300ND ND ND ND WWW 300 ND ND ND ND XXX 300 ND ND ND ND

Example 153 Synchronization of Rb-Positive Cells

HS68 cells were seeded out at 40,000 cells/well in 60 mm dish on day 1in DMEM containing 10% fetal bovine serum, 100 U/mlpenicillin/streptomycin and 1× Glutamax (Invitrogen) as described(Brookes et al. EMBO J, 21(12)2936-2945 (2002) and Ruas et al. Mol CellBiol, 27(12)4273-4282 (2007)). 24 hrs post seeding, cells are treatedwith compound T, compound Q, compound GG, compound U, PD0332991, or DMSOvehicle alone at 300 nM final concentration of test compounds. On day 3,one set of treated cell samples were harvested in triplicate (0 Hoursample). Remaining cells were washed two times in PBS-CMF and returnedto culture media lacking test compound. Sets of samples were harvestedin triplicate at 24, 40, and 48 hours.

Alternatively, the same experiment was done using normal Renal ProximalTubule Epithelial Cells (Rb-positive) obtained from American TypeCulture Collection (ATCC, Manassas, Va.). Cells were grown in anincubator at 37° C. in a humidified atmosphere of 5% CO2 in RenalEpithelial Cell Basal Media (ATCC) supplemented with Renal EpithelialCell Growth Kit (ATCC) in 37° C. humidified incubator.

Upon harvesting cells, samples were stained with propidium iodidestaining solution and samples run on Dako Cyan Flow Cytometer. Thefraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phaseDNA cell cycle was determined using FlowJo 7.2.2 analysis.

FIG. 1 shows cellular wash-out experiments which demonstrate theinhibitor compounds of the present invention have a short, transientG1-arresting effect in different cell types. Compounds T, Q, GG, and Uwere compared to PD0332991 in either human fibroblast cells(Rb-positive) (FIGS. 1A & 1B) or human renal proximal tubule epithelialcells (Rb-positive) (FIGS. 1C & 1D) and the effect on cell cyclefollowing washing out of the compounds was determined at 24, 36, 40, and48 hours.

As shown in FIG. 1 , using CDK4/6 inhibitors as described hereinprovides the ability to synchronize Rb-positive cells.

Example 154 Cell Cycle Arrest by Compound T in CDK4/6-Dependent Cells

To test the ability of CDK4/6 inhibitors to induce a clean G1-arrest inRb-positive cells, a cell based screening method was used consisting oftwo CDK4/6-dependent cell lines (tHS68 and WM2664; Rb-positive) and oneCDK4/6-independent (A2058; Rb-negative) cell line. Twenty-four hoursafter plating, each cell line was treated with Compound T in a dosedependent manner for 24 hours. At the conclusion of the experiment,cells were harvested, fixed, and stained with propidium iodide (a DNAintercalator), which fluoresces strongly red (emission maximum 637 nm)when excited by 488 nm light. Samples were run on Dako Cyan flowcytometer and >10,000 events were collected for each sample. Data wereanalyzed using FlowJo 2.2 software developed by TreeStar, Inc.

In FIG. 10A, results show that Compound T induces a robust G1 cell cyclearrest, as nearly all cells are found in the G0-G1 phase upon treatmentwith increasing amounts of Compound T. In FIG. 10A, the results showthat in CDK4/6-dependent cell lines, Compound T induced a robust G1 cellcycle arrest with an EC₅₀ of 80 nM in tHS68 cells with a correspondingreduction in S-phase ranging from 28% at baseline to 6% at the highestconcentration shown. Upon treatment with Compound T (300 nM), there wasa similar reduction in the S-phase population and an increase inG1-arrested cells in both CDK4/6-dependent cell lines (tHS68 (CompareFIGS. 10B and 10E) and WM2664 (Compare FIGS. 10C and 10F)), but not inthe CDK4/6-independent (A2058; Compare FIGS. 10D and 10G) cell line. TheCDK4/6-independent cell line shows no effect in the presence ofinhibitor.

Example 155 Compound T Inhibits Phosphorylation of RB

The CDK4/6-cyclin D complex is essential for progression from G1 to theS-phase of the DNA cell cycle. This complex phosphorylates theretinoblastoma tumor suppressor protein (Rb). To demonstrate the impactof CDK4/6 inhibition on Rb phosphorylation (pRb), Compound T was exposedto three cell lines, two CDK4/6 dependent (tHS68, WM2664; Rb-positive)and one CDK4/6 independent (A2058; Rb-negative). Twenty four hours afterseeding, cells were treated with Compound T at 300 nM finalconcentration for 4, 8, 16, and 24 hours. Samples were lysed and proteinwas assayed by western blot analysis. Rb phosphorylation was measured attwo sites targeted by the CDK4/6-cyclin D complex, Ser780 and Ser807/811using species specific antibodies. Results demonstrate that Compound Tblocks Rb phosphorylation in Rb-dependent cell lines by 16 hours postexposure, while having no effect on Rb-independent cells (FIG. 11 ).

Example 156 Differential Effects of a CDK4/6 Inhibitor on HematopoieticCells

Healthy C57BL/6 female mice received Compound T (50 mg or 100 mg) orvehicle control by i.p. injection. At the designated time: 30 min, 1 h,2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h or 48 h, mice received an injectionof 5′-ethynyl-2′-deoxyuridine (EdU). Two hours post EdU injection, micewere sacrificed. As illustrated in FIGS. 12-19 , cell lines wereisolated and incorporation of EdU was measured. Hematopoietic celllineages were identified using known cell lineage markers. (See, forexample, Challen, et al. “Mouse Hematopoietic Stem Cell Identificationand Analysis”, Cytometry A. (2009) 75(1): 14-24).

As evidence by FIGS. 12-19 , hematopoietic cells are differentiallyinhibited by the same dose of CDK4/6 inhibitor based on cell-type. Thedifferent cell types examined include bone marrow cells, thymocytes,myeloid cells (Mac1+/Gr1+), B lymphoid cells (B220+), erythroid cells(Ter119+), myeloid progenitor cells (LK+: Lin− Sca-1− c-kit+), andhematopoietic stem cells (LSK+: Lin− Sca-1+ c-kit+). This differentialinhibitory effect can be utilized to adjust the dose of the CDK4/6inhibitor used during a treatment strategy to provide for inhibition ofcertain lineages, for example diseased hematological lineages, while notinhibiting others.

Example 157 Enhanced Efficacy of a PI3 Kinase Inhibitor in Combinationwith a CDK 4/6 Inhibitor in a Mouse Model of Breast Cancer

The potential advantageous, additive, or synergistic effect of a PI3kinase inhibitor (pictilisib; GDC-0941) in combination with a CDK 4/6inhibitor (Compound GG) was examined in a mouse model of breast cancer.Immunodeficient mice were implanted with the human breast cancer cellline MCF7 (an Rb-positive cell line). Once the tumors reached sufficientsize, the mice were randomized into treatment cohorts as outlined inTable 5.

TABLE 5 Cohort Table Subjects Dose Group (N) Agents (mg/kg/dose)Route/Schedule 1 10 Vehicle — PO/QDX28 2 10 Compound GG 100 PO/QDXD1-D283 10 Compound GG 50 PO/QDXD1-D28 GDC-0941 100 PO/QDXD15-D28 4 10Compound GG 10 PO/QDXD1-D28 GDC-0941 100 PO/QDXD15-D28 5 10 GDC-0941 100IP/QDXD1-D14 PO/QDXD15-D28 6 10 Compound GG 100 PO/QDX28 GDC-0941 100IP/QDXD1-D14 PO/QDXD15-D28 7 10 PD0332991 100 PO/QDX28

Mice were treated with vehicle control (days 1-28) (group 1), 100 mg/kgCompound GG (days 1-28) (group 2), 50 mg/kg Compound GG (days 1-28) and100 mg/kg GDC-0941 (days 15-28) (group 3), 10 mg/kg Compound GG (days1-28) and 100 mg/kg GDC-0941 (days 15-28) (group 4), 100 mg/kg GDC-0941(IP dose days 1-14; PO dose days 15-28) (group 5), or 100 mg/kg CompoundGG (days 1-28) and 100 mg/kg GDC-0941 (IP dose days 1-14; PO dose days15-28) (group 6), and 100 mg/kg palbociclib (PD0332991) (days 1-28)(group 7). Compound GG was administered immediately prior toadministration of GDC-0941. Tumors were measured twice a week for 60days post treatment or until tumor burden endpoint was reached.

As seen in FIG. 20 , mice treated with a combination of 100 mg/kgCompound GG and 100 mg/kg GDC-0941 (pictilisib) for 28 days showedenhanced efficacy toward the MCF7 breast cancer cell line implants, ascompared to mice treated with vehicle control, 100 mg/kg Compound GG,100 mg/kg palbociclib, 50 mg/kg Compound GG and 100 mg/kg GDC-0941, 10mg/kg Compound GG and 100 mg/kg GDC-0941, or 100 mg/kg GDC-0941. Asshown in FIG. 21 , peripheral blood from mice in compound GG cohorts wasobtained on day 14 and CBCs were analyzed. Specific cell counts analyzedwere white blood cells (WBC: FIG. 21A), lymphocytes (FIG. 21B),neutrophils (FIG. 21C), monocytes (FIG. 21D), platelets (FIG. 21E), andred blood cells (FIG. 21F). As shown in FIG. 21 , white blood cells,lymphocytes, neutrophils, platelets, and red blood cells showed reducedcell counts on Day 14 in mice treated with 100 mg/kg Compound GG.

This specification has been described with reference to embodiments ofthe invention. The invention has been described with reference toassorted embodiments, which are illustrated by the accompanyingExamples. The invention can, however, be embodied in different forms andshould not be construed as limited to the embodiments set forth herein.Given the teaching herein, one of ordinary skill in the art will be ableto modify the invention for a desired purpose and such variations areconsidered within the scope of the invention.

We claim:
 1. A method for the treatment of an Rb-positive cancer in ahuman in need thereof comprising administering an effective amount ofBruton's tyrosine kinase (BTK) inhibitor selected from ibrutinib andacalabrutinib in combination with a CDK 4/6 inhibitor compound of thestructure

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the Rb-positive cancer is a solid tumor.
 3. The method of claim2, wherein the solid tumor is selected from non-small cell lung cancer,colorectal cancer, liver cancer, ovarian cancer, glioblastoma, breastcancer.
 4. The method of claim 3, wherein the solid tumor is non-smallcell lung cancer.
 5. The method of claim 3, wherein the solid tumor iscolorectal cancer.
 6. The method of claim 3, wherein the solid tumor isliver cancer.
 7. The method of claim 3, wherein the solid tumor isovarian cancer.
 8. The method of claim 3, wherein the solid tumor isglioblastoma.
 9. The method of claim 3, wherein the solid tumor isbreast cancer.
 10. The method of claim 9, wherein the breast cancer isErb-2/human epidermal growth factor receptor (HER)-2-positive breastcancer.
 11. The method of any of claim 1, wherein the Rb-positive canceris a hematological cancer.
 12. The method of claim 11, wherein thehematological cancer is acute myeloid leukemia (AML).
 13. The method ofclaim 11, wherein the hematological cancer is acute lymphocytic leukemia(ALL).
 14. The method of claim 11, wherein the hematological cancer is aT-cell cancer.
 15. The method of any of claim 1, wherein the CDK 4/6inhibitor compound is conjugated to a radioisotope.
 16. The method ofclaim 1, wherein the CDK 4/6 inhibitor compound is conjugated to atargeting agent.
 17. The method of claim 16, wherein the targeting agentis an antibody or antibody fragment.